Stabilized soluble prefusion rsv f polypeptides

ABSTRACT

Described are stable pre-fusion respiratory syncitial virus (RSV) F polypeptides, immunogenic compositions comprising the polypeptides and uses thereof for the prevention and/or treatment of RSV infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2014/058353, filed Apr. 24, 2014, designating the United States of America and published in English as International Patent Publication WO 2014/174018 A1 on Oct. 30, 2014, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 13165402.2, filed Apr. 25, 2013.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) OR (e) Sequence Listing Submitted as a TXT and PDF Files

Pursuant to 37 C.F.R. §1.821(c) or (e), files containing a TXT version and a PDF version of the Sequence Listing have been submitted concomitant with this application, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD

This disclosure relates to the field of medicine. The disclosure, in particular, relates to recombinant pre-fusion RSV F polypeptides and uses thereof, e.g., in immunogenic compositions.

BACKGROUND

Respiratory syncytial virus (RSV) is an enveloped non-segmented negative-strand RNA virus in the family Paramyxoviridae, genus Pneumovirus. Worldwide, it is estimated that 64 million RSV infections occur each year, resulting in 160,000 deaths (WHO Acute Respiratory Infections Update September 2009). The most severe disease occurs particularly in premature infants, the elderly and immune-compromised individuals. In children younger than 2 years, RSV is the most common respiratory tract pathogen, accounting for approximately 50% of the hospitalizations due to respiratory infections, with a peak of hospitalization occurring at 2-4 months of age. It has been reported that almost all children have been infected by RSV by the age of two. Repeated infection during lifetime is attributed to ineffective natural immunity. The level of RSV disease burden, mortality and morbidity in the elderly are second only to those caused by non-pandemic influenza A infections.

To infect a host cell, RSV, like other enveloped viruses such as influenza virus and HIV, require fusion of the viral membrane with a host cell membrane. For RSV, the conserved fusion protein (RSV F protein) fuses the viral and host cell cellular membranes. In current models, based on paramyxovirus studies, the RSV F protein initially folds into a “pre-fusion” conformation. During cell entry, the pre-fusion conformation undergoes refolding and conformational changes to its “post-fusion” conformation. Thus, the RSV F protein is a metastable protein that drives membrane fusion by coupling irreversible protein refolding to membrane juxtaposition by initially folding into a metastable form (pre-fusion conformation) that subsequently undergoes discrete/stepwise conformational changes to a lower energy conformation (post-fusion conformation).

It is clear from electron microscopy of RSV-F that large structural differences between the pre-fusion and post-fusion F trimer exist, which has recently been confirmed by crystallography (McLellan J. S. et al., Science 340(6136):1113-7 (2013) and McLellan J. S. et al., Science 342(6158):592-8 (2013)). These observations suggest that pre-fusion and post-fusion RSV F protein are antigenically distinct (Calder, L. J. et al., Virology 271:122-131 (2000)).

A vaccine against RSV infection is not currently available, but is desired. Vaccine candidates based on the RSV F protein have failed due to problems with, e.g., stability, purity, reproducibility, and potency. As indicated above, crystal structures have revealed a large conformational change between the pre-fusion and post-fusion states. The magnitude of the rearrangement suggested that only a portion of antibodies directed to the post-fusion conformation of RSV-F will be able to cross-react with the native conformation of the pre-fusion spike on the surface of the virus. Accordingly, efforts to produce a vaccine against RSV have focused on developing vaccines that contain pre-fusion forms of RSV F protein (see, e.g., WO2010/149743, WO2009/079796, and WO2012/158613). However, these efforts have not yet yielded stable pre-fusion RSV F polypeptides that could be used as candidates for testing in humans.

BRIEF SUMMARY

Provided are stable, recombinant, pre-fusion respiratory syncytial virus (RSV) fusion (F) polypeptides, i.e., recombinant RSV F polypeptides that are stabilized in the pre-fusion conformation. The RSV F polypeptides of the disclosure comprise at least one epitope that is specific to the pre-fusion conformation F protein. In certain embodiments, the pre-fusion RSV F polypeptides are soluble. In certain embodiments, the polypeptides are membrane-bound. Also provided are nucleic acid molecules (polynucleotides) encoding the pre-fusion RSV F polypeptides according to the disclosure and vectors comprising such nucleic acid molecules.

The disclosure also relates to compositions, preferably immunogenic compositions, comprising an RSV F polypeptide, a nucleic acid molecule and/or a vector, and to the use thereof in inducing an immune response against RSV F protein, in particular, the use thereof as a vaccine. The disclosure also relates to methods for inducing an anti-respiratory syncytial virus (RSV) immune response in a subject, comprising administering to the subject an effective amount of a pre-fusion RSV F polypeptide, a nucleic acid molecule encoding the RSV F polypeptide, and/or a vector comprising the nucleic acid molecule. Preferably, the induced immune response is characterized by neutralizing antibodies to RSV and/or protective immunity against RSV. In particular aspects, the disclosure relates to a method for inducing neutralizing anti-respiratory syncytial virus (RSV) F protein antibodies in a subject, comprising administering to the subject an effective amount of an immunogenic composition comprising a pre-fusion RSV F polypeptide, a nucleic acid molecule encoding the RSV F polypeptide, and/or a vector comprising the nucleic acid molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: FIG. 1A, SUPERDEX® 200 gel filtration chromatogram of the eluate A2_F24 N67I+S215P from the ion-exchange column. The arrows indicate the elution points of standard protein (1-Thyroglobulin 669 kDa, 2-Ferritin 440 kDa and 3-IgG 150 kDa). FIG. 1B, SDS-PAGE analysis of the pre-fusion F protein containing peak from the SEC chromatogram under reducing conditions.

FIG. 2: Western blot of NativePAGE loaded with samples containing 1) supernatant from cells expressing pre-fusion construct with the isoleucine zipper (S) F43; 2) supernatant from cells expressing mainly trimeric (top band) post-fusion RSV F protein; and 3) purified trimeric pre-fusion A2_F24 N67I.

FIG. 3: Expression levels of the point-mutated constructs relative to the non-mutated A2_F24.

FIGS. 4A and 4B: shows the results of the method described in Example 6; FIG. 4A illustrates determining the temperature where 50% of the CR9501 binding is lost; FIG. 4B shows a comparison of the stability of pre-fusion F (A2_F24 N67I+S215P) and the unmodified ectodomain when assessed by 50% loss of binding of the pre-fusion-specific antibody CR9501.

FIGS. 5A-5E: OCTET® measurements showing the storage-time-dependent loss of binding of the pre-fusion-specific antibody CR9501 to the pre-fusion constructs; FIG. 5A, A2_F24 (SEQ ID NO:19); FIG. 5B, A2_F24 K465Q; FIG. 5C, A2_F24 S46G; FIG. 5D, A2_F24 N67I; and FIG. 5E, A2_F24 E92D at days 1, 5 and 33.

FIGS. 6A-6I: OCTET® measurements showing the storage-time-dependent loss of binding of the pre-fusion-specific monoclonal antibody CR9501 to the pre-fusion constructs; FIG. 6A, A2_F24 K465Q; FIG. 6B, A2_F24 K465Q+N67I; FIG. 6C, A2_F24 S46G; FIG. 6D, A2_F24 S46G+E92D; FIG. 6E, A2_F24 S46G+N67I; FIG. 6F, A2_F24 E92D; FIG. 6G, A2_F24 S46G+E92D; FIG. 6H, A2_F24 N67I+E92D; and FIG. 6I, A2_F24 E92D+S215P at days 1, 5 and 33.

FIG. 7: VNA titers of mice at week 6 after a prime boost at week 0 and 4 with immunogens and doses according to Table 14.

FIG. 8: VNA titers of cotton rats at week 7 after a prime boost at week 0 and 4 with immunogens and doses according to Table 15.

FIG. 9: Lung and nose viral load at 5 days after i.n. RSV challenge.

DETAILED DESCRIPTION

The fusion protein (F) of the respiratory syncytial virus (RSV) is involved in fusion of the viral membrane with a host cell membrane, which is required for infection. The RSV F mRNA is translated into a 574-amino acid precursor protein designated F0, which contains a signal peptide sequence at the N-terminus (e.g., amino acid residues 1-26 of SEQ ID NO:1) that is removed by a signal peptidase in the endoplasmic reticulum. F0 is cleaved at two sites (between amino acid residues 109/110 and 136/137) by cellular proteases (in particular, furin) in the trans-Golgi, removing a short glycosylated intervening sequence (also referred to as a “p27 region,” comprising the amino acid residues 110 to 136, and generating two domains or subunits designated F1 and F2. The F1 domain (amino acid residues 137-574) contains a hydrophobic fusion peptide at its N-terminus and the C-terminus contains the transmembrane (TM) (amino acid residues 530-550) and cytoplasmic region (amino acid residues 551-574). The F2 domain (amino acid residues 27-109) is covalently linked to F1 by two disulfide bridges. The F1-F2 heterodimers are assembled as homotrimers in the virion.

A vaccine against RSV infection is not currently available, but is desired. One potential approach to producing a vaccine is a subunit vaccine based on purified RSV F protein. However, for this approach, it is desirable that the purified RSV F protein is in a conformation that resembles the conformation of the pre-fusion state of RSV F protein, that is stable over time, and can be produced in sufficient quantities. In addition, for a subunit-based vaccine, the RSV F protein needs to be truncated by deletion of the transmembrane (TM) and the cytoplasmic region to create a soluble secreted F protein (sF). Because the (TM) region is responsible for membrane anchoring and trimerization, the anchorless soluble F protein is considerably more labile than the full-length protein and will readily refold into the post-fusion end-state. In order to obtain soluble F protein in the stable pre-fusion conformation that shows high expression levels and high stability, the pre-fusion conformation thus needs to be stabilized.

Stabilization of another paramyxovirus F protein in the pre-fusion conformation has been successfully accomplished for parainfluenza type 5 (PIV-5). Yin et al. (Nature 439:38-44 (2006)) thus stabilized the pre-fusion structure of PIV-5 F protein by mutation of the furin cleavage site in F₀, which blocked processing into F1 and F2. Furthermore, the transmembrane (TM) and cytoplasmic domain were replaced by a well-known helical trimerization domain: GCN4-pII. This domain forms a trimeric helical coiled coil structure and is a modification of the natural dimeric helical coiled coil peptide GCN4 (O'Shea et al., Science 243:538-542 (1989)). The GCN4-pII peptide, in which the amino acid sequence of the GCN4 Leucine zipper was substituted with Isoleucine residues at every a and d position of the heptad, was shown to form a triple-stranded parallel alpha-helical coiled coil (Harbury et al., Science 262:1401-1407 (1993)).

For the stabilization of RSV F in the pre-fusion conformation, the same strategy has been tried, such as, e.g., mutation of the furin cleavage site, and fusion of the RSV-F ectodomain to a GCN4-pII trimerization domain (as disclosed in, e.g., WO2010/149743, WO2010/149745, WO2009/079796, and WO2012/158613) or to the fibritin trimerization domain (MCLellan et al., Nature Struct. Biol. 17:2-248-250 (2010)). This fibritin domain or “foldon” is derived from T4 fibritin and was described earlier as an artificial natural trimerization domain (Letarov et al., Biochemistry Moscow 64:817-823 (1993); S-Guthe et al., J. Mol. Biol. 337:905-915 (2004)). However, these efforts did not result in stable pre-fusion RSV-F protein. Moreover, these efforts have not yet resulted in candidates suitable for testing in humans.

This disclosure now provides recombinant stable pre-fusion RSV F polypeptides, i.e., RSV F polypeptides that are stabilized in the pre-fusion conformation. In the research that led to this disclosure, several modification steps were introduced and/or combined in order to obtain the stable soluble pre-fusion RSV F polypeptides. The stable pre-fusion RSV F polypeptides of the disclosure are in the pre-fusion conformation, i.e., they comprise (display) at least one epitope that is specific to the pre-fusion conformation F protein. An epitope that is specific to the pre-fusion conformation F protein is an epitope that is not presented in the post-fusion conformation. Without wishing to be bound by any particular theory, it is believed that the pre-fusion conformation of RSV F protein may contain epitopes that are the same as those on the RSV F protein expressed on natural RSV virions and, therefore, may provide advantages for eliciting protective neutralizing antibodies.

In certain embodiments, the polypeptides of the disclosure comprise at least one epitope that is recognized by a pre-fusion specific monoclonal antibody, comprising a heavy chain CDR1 region of SEQ ID NO:54, a heavy chain CDR2 region of SEQ ID NO:55, a heavy chain CDR3 region of SEQ ID NO:56 and a light chain CDR1 region of SEQ ID NO:62, a light chain CDR2 region of SEQ ID NO:63, and a light chain CDR3 region of SEQ ID NO:64 (hereafter referred to as CR9501) and/or a pre-fusion specific monoclonal antibody, comprising a heavy chain CDR1 region of SEQ ID NO:58, a heavy chain CDR2 region of SEQ ID NO:59, a heavy chain CDR3 region of SEQ ID NO:60 and a light chain CDR1 region of SEQ ID NO:66, a light chain CDR2 region of SEQ ID NO:67, and a light chain CDR3 region of SEQ ID NO:68 (referred to as CR9502). CR9501 and CR9502 comprise the heavy and light chain variable regions and, thus, the binding specificities of the antibodies 58C5 and 30D8, respectively, which have previously been shown to bind specifically to RSV F protein in its pre-fusion conformation and not to the post-fusion conformation (see WO2012/006596).

In certain embodiments, the recombinant pre-fusion RSV F polypeptides comprise at least one epitope that is recognized by at least one pre-fusion specific monoclonal antibody as described above and are trimeric.

In certain embodiments, the stable pre-fusion RSV F polypeptides according to the disclosure comprise a mutation of the amino acid residue on position 67 and/or a mutation of the amino acid residue on position 215.

In certain embodiments, the amino acid on position 67 is mutated to a hydrophobic amino acid.

In certain embodiments, the stable pre-fusion RSV F polypeptides according to the disclosure comprise a mutation of the amino acid residue N or T on position 67 and/or a mutation of amino acid residue S on position 215.

In certain embodiments, the stable pre-fusion RSV F polypeptides according to the disclosure comprise an F1 domain and an F2 domain, and a linking sequence comprising from 1 to 10 amino acid residues, linking the F1 domain to the F2 domain, wherein the polypeptides further comprise a mutation of the amino acid residue N or T on position 67 and/or a mutation of amino acid residue S on position 215.

In certain embodiments, the stable pre-fusion RSV F polypeptides according to the disclosure comprise a truncated F1 domain and an F2 domain, and a linking sequence comprising from 1 to 10 amino acid residues, linking the truncated F1 domain to the F2 domain, wherein the polypeptides further comprise a mutation of the amino acid residue N or T on position 67 and/or a mutation of amino acid residue S on position 215.

The polypeptides of the disclosure thus comprise at least one stabilizing mutation in the F1 and/or F2 domain as compared to the RSV F1 and/or F2 domain in a wild-type RSV F protein.

In certain embodiments, the pre-fusion RSV F polypeptides comprise a mutation of amino acid residue N or T on position 67 (N/T67I) into I and/or a mutation of amino acid residue S on position 215 into P (S215P).

It is known that RSV exists as a single serotype having two antigenic subgroups: A and B. The amino acid sequences of the mature processed F proteins of the two groups are about 93% identical. As used throughout the present application, the amino acid positions are given in reference to the sequence of RSV F protein from the A2 strain (SEQ ID NO:1). As used in this disclosure, the wording “the amino acid at position “x” of the RSV F protein” thus means the amino acid corresponding to the amino acid at position “x” in the RSV F protein of the RSV A2 strain of SEQ ID NO:1. Note that, in the numbering system used throughout this application, “1” refers to the N-terminal amino acid of an immature F0 protein (SEQ ID NO:1). When an RSV strain other than the A2 strain is used, the amino acid positions of the F protein are to be numbered with reference to the numbering of the F protein of the A2 strain of SEQ ID NO:1 by aligning the sequences of the other RSV strain with the F protein of SEQ ID NO:1 with the insertion of gaps as needed. Sequence alignments can be done using methods well known in the art, e.g., by CLUSTALW, Bioedit or CLC Workbench.

An amino acid according to the disclosure can be any of the twenty naturally occurring (or “standard” amino acids) or variants thereof, such as, e.g., D-amino acids (the D-enantiomers of amino acids with a chiral center), or any variants that are not naturally found in proteins, such as, e.g., norleucine. The standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties such as cysteine, that can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, proline that induces turns of the polypeptide backbone, and glycine that is more flexible than other amino acids. Table 11 shows the abbreviations and properties of the standard amino acids.

It will be appreciated by a skilled person that the mutations can be made to the protein by routine molecular biology procedures. The mutations according to the disclosure preferably result in increased expression levels and/or increased stabilization of the pre-fusion RSV F polypeptides as compared to RSV F polypeptides that do not comprise these mutation(s).

In certain embodiments, the pre-fusion RSV F polypeptides are soluble.

In certain embodiments, the pre-fusion RSV F polypeptides further comprise a heterologous trimerization domain linked to the truncated F1 domain. According to the disclosure, it was shown that by linking a heterologous trimerization domain to the C-terminal amino acid residue of a truncated F1 domain, optionally combined with a linking sequence linking the F1 and F2 domains, and the stabilizing mutation(s), RSV F polypeptides are provided that show high expression and that bind to pre-fusion-specific antibodies, indicating that the polypeptides are in the pre-fusion conformation. In addition, the RSV F polypeptides are stabilized in the pre-fusion conformation, i.e., even after processing of the polypeptides, they still bind to the pre-fusion-specific antibodies CR9501 and/or CR9502, indicating that the pre-fusion-specific epitope is retained.

In further embodiments, the pre-fusion RSV F polypeptides comprise one or more further mutations (as compared to the wild-type RSV F protein), selected from the group consisting of:

-   -   (a) a mutation of the amino acid residue on position 46;     -   (b) a mutation of the amino acid residue on position 77;     -   (c) a mutation of the amino acid residue on position 80;     -   (d) a mutation of the amino acid residue on position 92;     -   (e) a mutation of the amino acid residue on position 175;     -   (f) a mutation of the amino acid residue on position 184;     -   (g) a mutation of the amino acid residue on position 185;     -   (h) a mutation of the amino acid residue on position 201;     -   (i) a mutation of the amino acid residue on position 209;     -   (j) a mutation of the amino acid residue on position 421;     -   (k) a mutation of the amino acid residue on position 426;     -   (l) a mutation of the amino acid residue on position 465;     -   (m) a mutation of the amino acid residue on position 486;     -   (n) a mutation of the amino acid residue on position 487; and     -   (o) a mutation of the amino acid residue on position 508.

In preferred embodiments, the one or more further mutations are selected from the group consisting of:

-   -   (a) a mutation of the amino acid residue S on position 46 into G         (S46G);     -   (b) a mutation of the amino acid residue K on position 77 into E         (K77E);     -   (c) a mutation of the amino acid residue K on position 80 into E         (K80E);     -   (d) a mutation of the amino acid residue E on position 92 into D         (E92D);     -   (e) a mutation of the amino acid residue N on position 175 into         P (N175P);     -   (f) a mutation of the amino acid residue G on position 184 into         N (G184N);     -   (g) a mutation of the amino acid residue V on position 185 into         N (V185N);     -   (h) a mutation of the amino acid residue K on position 201 into         Q (K201Q);     -   (i) a mutation of the amino acid residue K on position 209 into         Q (K209Q);     -   (j) a mutation of the amino acid residue K on position 421 into         N (K421N);     -   (k) a mutation of the amino acid residue N on position 426 into         S (N426S);     -   (l) a mutation of the amino acid residue K on position 465 into         E or Q (K465E/Q);     -   (m) a mutation of the amino acid residue D on position 486 into         N (D486N);     -   (n) a mutation of the amino acid residue E on position 487 into         Q, N or I (E487Q/N/I); and     -   (o) a mutation of the amino acid residue K on position 508 into         E (K508E).

It is again noted that for the positions of the amino acid residues, reference is made to SEQ ID NO:1. A skilled person will be able to determine the corresponding amino acid residues in F proteins of other RSV strains.

In certain embodiments, the pre-fusion RSV F polypeptides comprise at least two mutations (as compared to a wild-type RSV F protein). In preferred embodiments, the at least two mutations are a mutation of the amino acid N or T on position 67 into I (N/T67I) and a mutation of the amino acid S on position 215 into P (S215P).

In certain embodiments, the pre-fusion RSV F polypeptides comprise at least one further mutation, selected from the group consisting of:

-   -   (a) a mutation of the amino acid residue S on position 46 into         G;     -   (b) a mutation of the amino acid residue K on position 77 into         E;     -   (c) a mutation of the amino acid residue K on position 80 into         E;     -   (d) a mutation of the amino acid residue E on position 92 into         D;     -   (e) a mutation of the amino acid residue N on position 175 into         P;     -   (f) a mutation of the amino acid residue G on position 184 into         N;     -   (g) a mutation of the amino acid residue V on position 185 into         N;     -   (h) a mutation of the amino acid residue K on position 201 into         Q;     -   (i) a mutation of the amino acid residue K on position 209 into         Q;     -   (j) a mutation of the amino acid residue K on position 421 into         N;     -   (k) a mutation of the amino acid residue N on position 426 into         S;     -   (l) a mutation of the amino acid residue K on position 465 into         E or Q;     -   (m) a mutation of the amino acid residue D on position 486 into         N;     -   (n) a mutation of the amino acid residue E on position 487 into         Q, N or I; and     -   (o) a mutation of the amino acid residue K or R on position 508         into E.

In certain embodiments, the polypeptides comprise at least three mutations.

In certain embodiments, the heterologous trimerization domain comprises the amino acid sequence EKKIEAIEKKIEAIEKKIEA (SEQ ID NO:3). In certain other embodiments, the heterologous trimerization domain comprises the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:4).

As described above, in certain embodiments the polypeptides of the disclosure comprise a truncated F1 domain. As used herein, a “truncated” F1 domain refers to an F1 domain that is not a full-length F1 domain, i.e., wherein, either N-terminally or C-terminally, one or more amino acid residues have been deleted. According to the disclosure, at least the transmembrane domain and cytoplasmic tail have been deleted to permit expression as a soluble ectodomain.

In certain other embodiments, the F1 domain is truncated after amino acid residue 495 of the RSV F protein (referring to SEQ ID NO:1), i.e., the C-terminal part of the F1 domain starting from amino acid residue 496 (referring to SEQ ID NO:1) has been deleted. In certain other embodiments, the F1 domain is truncated after amino acid residue 513 of the RSV F protein. In certain embodiments, the F1 domain is truncated after amino acid residue 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524 or 525.

In certain embodiments, the trimerization domain is linked to amino acid residue 495 of the RSV F1 domain. In certain embodiments, the trimerization domain comprises SEQ ID NO:4 and is linked to amino acid residue 495 of the RSV F1 domain.

In certain other embodiments, the trimerization domain is linked to amino acid residue 513 of the RSV F1 domain. In certain embodiments, the trimerization domain comprises SEQ ID NO:3 and is linked to amino acid residue 513 of the RSV F1 domain.

In certain embodiments, the F1 domain, which is optionally truncated, and the F2 domain are linked by a linking sequence, linking the C-terminal amino acid of the F2 domain to the N-terminal amino acid of the (optionally truncated) F1 domain. In certain embodiments, the linking sequence (or linker) comprises from 1-10 amino acid residues, preferable from 2-9 amino acid residues, preferably from 3-8 amino acid residues, preferably from 4-7 amino acid residues, more preferably, the linker comprises 5 or 6 amino acid residues. Numerous conformationally neutral linkers are known in the art that can be used according to the disclosure without disrupting the conformation of the pre-fusion RSV F polypeptides. In preferred embodiments, the linker comprises the amino acid sequence GSGSG (SEQ ID NO:5).

In certain embodiments, the F1 domain and/or the F2 domain are from an RSV A strain. In certain embodiments, the F1 and/or F2 domain are from the RSV A2 strain of SEQ ID NO:1.

In certain embodiments, the F1 domain and/or the F2 domain are from an RSV A strain, such as from the RSV A strain of SEQ ID NO:69.

In certain embodiments, the F1 domain and/or the F2 domain are from an RSV B strain. In certain embodiments, the F1 and/or F2 domain are from the RSV B strain of SEQ ID NO:2.

In certain embodiments, the F1 and F2 domain are from the same RSV strain. In certain embodiments, the pre-fusion RSV F polypeptides are chimeric polypeptides, i.e., comprising F1 and F2 domains that are from different RSV strains.

In certain embodiments, the level of expression of the pre-fusion RSV F polypeptides of the disclosure is increased, as compared to a wild-type RSV F polypeptide ectodomain (i.e., without the transmembrane and cytoplasmic region) without the mutation(s). In certain embodiments, the level of expression is increased at least five-fold, preferably up to ten-fold. In certain embodiments, the level of expression is increased more than ten-fold.

The pre-fusion RSV F polypeptides according to the disclosure are stable, i.e., do not readily change into the post-fusion conformation upon processing of the polypeptides, such as, e.g., purification, freeze-thaw cycles, and/or storage, etc.

In certain embodiments, the pre-fusion RSV F polypeptides according to the disclosure have an increased stability upon storage at 4° C., as compared to an RSV F polypeptide without the mutation(s). In certain embodiments, the polypeptides are stable upon storage at 4° C. for at least 30 days, preferably at least 60 days, preferably at least 6 months, even more preferably at least 1 year. With “stable upon storage,” it is meant that the polypeptides still display the at least one epitope specific for the pre-fusion specific antibody (e.g., CR9501) upon storage of the polypeptide in solution (e.g., culture medium) at 4° C. for at least 30 days, e.g., as determined using a method as described in Example 7 or 9. In certain embodiments, the polypeptides display the at least one pre-fusion-specific epitope for at least 6 months, preferably for at least 1 year upon storage of the pre-fusion RSV F polypeptides at 4° C.

In certain embodiments, the pre-fusion RSV F polypeptides according to the disclosure have an increased stability when subjected to heat, as compared to RSV F polypeptides without the mutation(s). In certain embodiments, the pre-fusion RSV F polypeptides are heat stable for at least 30 minutes at a temperature of 55° C., preferably at 58° C., more preferably at 60° C. With “heat stable” it is meant that the polypeptides still display the at least one pre-fusion-specific epitope after having been subjected for at least 30 minutes to an increased temperature (i.e., a temperature of 55° C. or above), e.g., as determined using a method as described in Example 6.

In certain embodiments, the polypeptides display the at least one pre-fusion-specific epitope after being subjected to 1 to 6 freeze-thaw cycles in an appropriate formulation buffer.

In certain preferred embodiments, the pre-fusion RSV F polypeptide of the disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:21-52 and 71-89. In certain embodiments, the pre-fusion RSV F polypeptide of the disclosure consists of an amino acid sequence selected from the group consisting of SEQ ID NOS:21-52 and 71-89.

As used throughout this disclosure, nucleotide sequences are provided from 5′ to 3′ direction, and amino acid sequences from N-terminus to C-terminus, as customary in the art.

In certain embodiments, the encoded polypeptides according to the disclosure further comprise a leader sequence, also referred to as “signal sequence” or “signal peptide,” corresponding to amino acids 1-26 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:69. This is a short (typically 5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined toward the secretory pathway. In certain embodiments, the polypeptides according to the disclosure do not comprise a leader sequence.

In certain embodiments, the polypeptides comprise a HIS-Tag. A His-Tag or polyhistidine-tag is an amino acid motif in proteins that consists of at least five histidine (H) residues, often at the N- or C-terminus of the protein, which is generally used for purification purposes.

In certain embodiments, the polypeptides do not comprise a HIS-Tag. According to the disclosure, it has surprisingly been shown that when the HIS-tag is deleted, the level of expression and the stability are increased as compared to polypeptides with a HIS-tag.

This disclosure further provides nucleic acid molecules encoding the RSV F polypeptides according to the disclosure.

In preferred embodiments, the nucleic acid molecules encoding the polypeptides according to the disclosure are codon-optimized for expression in mammalian cells, preferably human cells. Methods of codon-optimization are known and have been described previously (e.g., WO 96/09378). A sequence is considered codon-optimized if at least one non-preferred codon as compared to a wild-type sequence is replaced by a codon that is more preferred. Herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non-preferred codon. The frequency of codon usage for a specific organism can be found in codon frequency tables, such as on the World Wide Web at kazusa.or.jp/codon. Preferably, more than one non-preferred codon, preferably most or all non-preferred codons, are replaced by codons that are more preferred. Preferably, the most frequently used codons in an organism are used in a codon-optimized sequence. Replacement by preferred codons generally leads to higher expression.

It will be understood by a skilled person that numerous different polynucleotides and nucleic acid molecules can encode the same polypeptide as a result of the degeneracy of the genetic code. It is also understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the nucleic acid molecules to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Therefore, unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may or may not include introns.

Nucleic acid sequences can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g., GENEART®, GENSCRIPT®, INVITROGEN®, and EUROFINS®).

The disclosure also provides vectors comprising a nucleic acid molecule as described above. In certain embodiments, a nucleic acid molecule according to the disclosure thus is part of a vector. Such vectors can easily be manipulated by methods well known to the person skilled in the art, and can, for instance, be designed for being capable of replication in prokaryotic and/or eukaryotic cells. In addition, many vectors can be used for transformation of eukaryotic cells and will integrate in whole or in part into the genome of such cells, resulting in stable host cells comprising the desired nucleic acid in their genome. The vector used can be any vector that is suitable for cloning DNA and that can be used for transcription of a nucleic acid of interest. Suitable vectors according to the disclosure are, e.g., adenovectors, such as, e.g., Ad26 or Ad35, alphavirus, paramyxovirus, vaccinia virus, herpes virus, retroviral vectors, etc. The person skilled in the art is capable of choosing suitable expression vectors, and inserting the nucleic acid sequences of the disclosure in a functional manner.

Host cells comprising the nucleic acid molecules encoding the pre-fusion RSV F polypeptides also form part of the disclosure. The pre-fusion RSV F polypeptides may be produced through recombinant DNA technology involving expression of the molecules in host cells, e.g., Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6® cells, or yeast, fungi, insect cells, and the like, or transgenic animals or plants. In certain embodiments, the cells are from a multicellular organism; in certain embodiments, they are of vertebrate or invertebrate origin. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human cells. In general, the production of recombinant proteins in a host cell, such as the pre-fusion RSV F polypeptides of the disclosure, comprises the introduction of a heterologous nucleic acid molecule encoding the polypeptide in expressible format into the host cell, culturing the cells under conditions conducive to expression of the nucleic acid molecule and allowing expression of the polypeptide in the cell. The nucleic acid molecule encoding a protein in expressible format may be in the form of an expression cassette, and usually requires sequences capable of bringing about expression of the nucleic acid, such as enhancer(s), promoter, polyadenylation signal, and the like. The person skilled in the art is aware that various promoters can be used to obtain expression of a gene in host cells. Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed.

Cell culture media are available from various vendors, and a suitable medium can be routinely chosen for a host cell to express the protein of interest, here, the pre-fusion RSV F polypeptides. The suitable medium may or may not contain serum.

A “heterologous nucleic acid molecule” (also referred to herein as “transgene”) is a nucleic acid molecule that is not naturally present in the host cell. It is introduced into, for instance, a vector by standard molecular biology techniques. A transgene is generally operably linked to expression control sequences. This can, for instance, be done by placing the nucleic acid encoding the transgene(s) under the control of a promoter. Further regulatory sequences may be added. Many promoters can be used for expression of a transgene(s), and are known to the skilled person, e.g., these may comprise viral, mammalian, synthetic promoters, and the like. A non-limiting example of a suitable promoter for obtaining expression in eukaryotic cells is a CMV-promoter (U.S. Pat. No. 5,385,839), e.g., the CMV immediate early promoter, for instance, comprising nt. −735 to +95 from the CMV immediate early gene enhancer/promoter. A polyadenylation signal, for example, the bovine growth hormone polyA signal (U.S. Pat. No. 5,122,458), may be present behind the transgene(s). Alternatively, several widely used expression vectors are available in the art and from commercial sources, e.g., the pcDNA and pEF vector series of INVITROGEN®, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from STRATAGENE™, etc., which can be used to recombinantly express the protein of interest, or to obtain suitable promoters and/or transcription terminator sequences, polyA sequences, and the like.

The cell culture can be any type of cell culture, including adherent cell culture, e.g., cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture. Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. Nowadays, continuous processes based on perfusion principles are becoming more common and are also suitable. Suitable culture media are also well known to the skilled person and can generally be obtained from commercial sources in large quantities, or custom-made according to standard protocols. Culturing can be done, for instance, in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems, and the like. Suitable conditions for culturing cells are known (see, e.g., Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and R. I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9)).

The disclosure further provides compositions comprising a pre-fusion RSV F polypeptide and/or a nucleic acid molecule, and/or a vector, as described above. The disclosure thus provides compositions comprising a pre-fusion RSV F polypeptide that displays an epitope that is present in a pre-fusion conformation of the RSV F protein but is absent in the post-fusion conformation. The disclosure also provides compositions comprising a nucleic acid molecule and/or a vector, encoding such pre-fusion RSV F polypeptide. The disclosure further provides immunogenic compositions comprising a pre-fusion RSV F polypeptide, and/or a nucleic acid molecule, and/or a vector, as described above. The disclosure also provides the use of a stabilized pre-fusion RSV F polypeptide, a nucleic acid molecule, and/or a vector, according to the disclosure, for inducing an immune response against RSV F protein in a subject. Further provided are methods for inducing an immune response against RSV F protein in a subject, comprising administering to the subject a pre-fusion RSV F polypeptide, and/or a nucleic acid molecule, and/or a vector, according to the disclosure. Also provided are pre-fusion RSV F polypeptides, nucleic acid molecules, and/or vectors, according to the disclosure for use in inducing an immune response against RSV F protein in a subject. Further provided is the use of the pre-fusion RSV F polypeptides, and/or nucleic acid molecules, and/or vectors according to the disclosure for the manufacture of a medicament for use in inducing an immune response against RSV F protein in a subject.

The pre-fusion RSV F polypeptides, nucleic acid molecules, or vectors of the disclosure may be used for prevention (prophylaxis) and/or treatment of RSV infections. In certain embodiments, the prevention and/or treatment may be targeted at patient groups that are susceptible to RSV infection. Such patient groups include, but are not limited to, e.g., the elderly (e.g., ≧50 years old, ≧60 years old, and preferably ≧65 years old), the young (e.g., ≦5 years old and ≦1 year old), hospitalized patients and patients who have been treated with an antiviral compound but have shown an inadequate antiviral response.

The pre-fusion RSV F polypeptides, nucleic acid molecules and/or vectors according to the disclosure may be used, e.g., in stand-alone treatment and/or prophylaxis of a disease or condition caused by RSV, or in combination with other prophylactic and/or therapeutic treatments, such as (existing or future) vaccines, antiviral agents and/or monoclonal antibodies.

The disclosure further provides methods for preventing and/or treating RSV infection in a subject utilizing the pre-fusion RSV F polypeptides, nucleic acid molecules and/or vectors according to the disclosure. In a specific embodiment, a method for preventing and/or treating RSV infection in a subject comprises administering to a subject in need thereof an effective amount of a pre-fusion RSV F polypeptide, nucleic acid molecule and/or a vector, as described above. A therapeutically effective amount refers to an amount of a polypeptide, nucleic acid molecule or vector that is effective for preventing, ameliorating and/or treating a disease or condition resulting from infection by RSV. Prevention encompasses inhibiting or reducing the spread of RSV or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection by RSV. “Amelioration” as used in herein may refer to the reduction of visible or perceptible disease symptoms, viremia, or any other measurable manifestation of influenza infection.

For administering to subjects, such as humans, the disclosure may employ pharmaceutical compositions comprising a pre-fusion RSV F polypeptide, a nucleic acid molecule and/or a vector as described herein, and a pharmaceutically acceptable carrier or excipient. In the present context, the term “pharmaceutically acceptable” means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]). The RSV F polypeptides, or nucleic acid molecules, preferably are formulated and administered as a sterile solution, although it may also be possible to utilize lyophilized preparations. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, e.g., pH 5.0 to 7.5. The RSV F polypeptides typically are in a solution having a suitable pharmaceutically acceptable buffer, and the composition may also contain a salt. Optionally, stabilizing agent may be present, such as albumin. In certain embodiments, detergent is added. In certain embodiments, the RSV F polypeptides may be formulated into an injectable preparation.

In certain embodiments, a composition according to the disclosure further comprises one or more adjuvants. Adjuvants are known in the art to further increase the immune response to an applied antigenic determinant. The terms “adjuvant” and “immune stimulant” are used interchangeably herein, and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to the RSV F polypeptides of the disclosure. Examples of suitable adjuvants include aluminium salts such as aluminium hydroxide and/or aluminium phosphate; oil-emulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see, e.g., WO 90/14837); saponin formulations, such as, for example, QS21 and Immunostimulating Complexes (ISCOMS) (see, e.g., U.S. Pat. No. 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as E. coli, heat labile enterotoxin LT, cholera toxin CT, and the like; eukaryotic proteins (e.g., antibodies or fragments thereof (e.g., directed against the antigen itself or CD1a, CD3, CD7, or CD80) and ligands to receptors (e.g., CD40L, GMCSF, GCSF, etc.), which stimulate immune response upon interaction with recipient cells. In certain embodiments, the compositions of the disclosure comprise aluminium as an adjuvant, e.g., in the form of aluminium hydroxide, aluminium phosphate, aluminium potassium phosphate, or combinations thereof, in concentrations of 0.05-5 mg, e.g., from 0.075-1.0 mg, of aluminium content per dose.

The pre-fusion RSV F polypeptides may also be administered in combination with or conjugated to nanoparticles, such as, e.g., polymers, liposomes, virosomes, and virus-like particles. The pre-fusion F polypeptides may be combined with, encapsidated in, or conjugated to the nanoparticles with or without adjuvant. Encapsulation within liposomes is described, e.g., in U.S. Pat. No. 4,235,877. Conjugation to macromolecules is disclosed, for example, in U.S. Pat. No. 4,372,945 or U.S. Pat. No. 4,474,757.

In other embodiments, the compositions do not comprise adjuvants.

In certain embodiments, the disclosure provides methods for making a vaccine against respiratory syncytial virus (RSV), comprising providing a composition according to the disclosure and formulating it into a pharmaceutically acceptable composition. The term “vaccine” refers to an agent or composition containing an active component effective to induce a certain degree of immunity in a subject against a certain pathogen or disease, which will result in at least a decrease (up to complete absence) of the severity, duration or other manifestation of symptoms associated with infection by the pathogen or the disease. In this disclosure, the vaccine comprises an effective amount of a pre-fusion RSV F polypeptide and/or a nucleic acid molecule encoding a pre-fusion RSV F polypeptide, and/or a vector comprising the nucleic acid molecule, which results in an immune response against the F protein of RSV. This provides a method of preventing serious lower respiratory tract disease leading to hospitalization and the decrease in frequency of complications such as pneumonia and bronchiolitis due to RSV infection and replication in a subject. The term “vaccine” according to the disclosure implies that it is a pharmaceutical composition and, thus, typically includes a pharmaceutically acceptable diluent, carrier or excipient. It may or may not comprise further active ingredients. In certain embodiments, it may be a combination vaccine that further comprises other components that induce an immune response, e.g., against other proteins of RSV and/or against other infectious agents. The administration of further active components may, for instance, be done by separate administration or by administering combination products of the vaccines of the disclosure and the further active components.

Compositions may be administered to a subject, e.g., a human subject. The total dose of the RSV F polypeptides in a composition for a single administration can, for instance, be about 0.01 μg to about 10 mg, e.g., 1 μg-1 mg, e.g., 10 μg-100 μg. Determining the recommended dose will be carried out by experimentation and is routine for those skilled in the art.

Administration of the compositions according to the disclosure can be performed using standard routes of administration. Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transcutaneous, or mucosal administration, e.g., intranasal, oral, and the like. In one embodiment, a composition is administered by intramuscular injection. The skilled person knows the various possibilities to administer a composition, e.g., a vaccine, in order to induce an immune response to the antigen(s) in the vaccine.

A “subject” as used herein, is preferably a mammal, for instance, a rodent, e.g., a mouse, a cotton rat, or a non-human-primate, or a human. Preferably, the subject is a human subject.

The polypeptides, nucleic acid molecules, vectors, and/or compositions may also be administered, either as prime, or as boost, in a homologous or heterologous prime-boost regimen. Typically, if a boosting vaccination is performed, such a boosting vaccination will be administered to the same subject at a time between one week and one year, preferably between two weeks and four months, after administering the composition to the subject for the first time (which is in such cases referred to as “priming vaccination”). In certain embodiments, the administration comprises a prime and at least one booster administration.

In addition, the polypeptides of the disclosure may be used as diagnostic tools, for example, to test the immune status of an individual by establishing whether there are antibodies in the serum of such individual capable of binding to the polypeptide of the disclosure. Thus, the disclosure also relates to an in vitro diagnostic method for detecting the presence of an RSV infection in a patient, the method comprising the steps of a) contacting a biological sample obtained from the patient with a polypeptide according to the disclosure; and b) detecting the presence of antibody-polypeptide complexes.

The disclosure further provides a method for stabilizing the pre-fusion conformation of an RSV F polypeptide, comprising introducing one or more mutations in an RSV F1 and/or F2 domain, as compared to the wild-type RSV F1 and/or F2 domain, wherein the one or more mutations are selected from the group consisting of:

-   -   (a) a stabilizing mutation that locks the HRA domain from         hinging in a region adjacent to the conserved 69-212 disulfide         bridge, the region comprising the amino acid residues 66-68 and         214-216,     -   (b) a mutation in the helix (at the C-terminus of the F2 domain)         comprising the amino acid residues 76-98 at the C-terminus of         the F2 domain;     -   (c) a mutation that reduces the negative charge repulsion         between the top of the HRB stem region (N-terminal end of HRB)         comprising amino acids 486, 487 and 489; and     -   (d) a stabilizing mutation in the HRA region.

In certain embodiments, the mutation in the HRA hinge region is at position 67.

In certain embodiments, the mutation in the HRA hinge region is at position 215.

In certain embodiments, the mutation in the HRA hinge region is at position 66 or 68, and/or at position 214 or 216.

In certain embodiments, the mutation in the helix is at position 77.

In certain embodiments, the mutation in the helix is at position 80.

In certain embodiments, the amino acid residue at position 77 and/or 80 is changed into a negatively charged amino acid.

In certain embodiments, the mutation is at position 92.

In certain embodiments, the mutation that reduces the negative charge repulsion between the top of the HRB stem region comprises amino acids 486, 487, and 489.

In certain embodiments, the mutation is at position 489.

In certain embodiments, the mutation is at position 486.

In certain embodiments, the mutation stabilizes the beta-turns between the amino acid residues 175-193.

In certain embodiments, the mutation is stabilizing the turn at position 175.

In certain embodiments, the mutation is stabilizing the turn at position 184 and 185.

Stabilized pre-fusion RSV F polypeptides obtainable and/or obtained by such method also form part of the disclosure, as well as the uses thereof as described above.

The disclosure is further explained in the examples below. The examples do not limit the disclosure in any way; they merely serve to clarify the disclosure.

EXAMPLES Example 1 Preparation of Stable Pre-Fusion RSV F Polypeptides—Linkers and Trimerization Domains

In the research that led to this disclosure, stabilized variants of soluble pre-fusion F protein (sF) were designed by stabilizing the two main regions that initiate refolding. The first strategy was to arrest the fusion peptide in its position and prevent it from getting released from the head region by fixing and joining the F1-F2 domains by a short loop. Release of the fusion peptide can be prevented by re-establishing a covalent connection of the N-terminus of F1 to the C-terminus of F2. As shown in this example, several different linkers have been tried. The insertion of a five-amino acid loop between F1 and F2, in particular, comprising the amino acid sequence GSGSG (SEQ ID NO:5), was most successful. This linker was designed based on the distances measured in a 3D homology model that was generated for RSV-F type A2 based on the sequence alignment with the F sequence of parainfluenza type 5 for which a three-dimensional structure is published (Yin et. al., 2006).

SEQ ID NO: 90 (first line) SEQ ID NO: 91 (second line) SEQ ID NO: 92 (third line) AAA47881PIV5 -------------MGTIIQFLVVSCLLAGAGSLDPAALMQIGVIPTNVRQLMYYTEASSA FUS_HRSV1B ------MELLIHRSSAIFLTLAVNALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYT ACO83301HRSVA2ref ------MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYT               .:*   *..  :   :..  .  : *    ...   :     .  : AAA47881PIV5 FIVVKLMPTIDSPISGCNI--TSISSYNATVTKLLQPIGENLETIRNQLIP--TRRRRR- FUS_HRSV1B SVITIELSNIKET--KCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAP ACO83301HRSVA2ref SVITIELSNIKKN--KCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELP  ::.  :..*..    **   :.:.  :  : *  :.: *    ::.   .    **. AAA47881PIV5 -----------------FAGVVIGLAALGVATAAQVTAAVALVKANENAAAILNLKNAIQ FUS_HRSV1B QYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALL ACO83301HRSVA2ref RFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALL                         : .: :...: :::.:*: *. .  . : ::*.*: AAA47881PIV5 KTNAAVADVVQATQSLGTAVQAVQDHINSVVSPAITAANCKAQDAIIGSILNLYLTELTT FUS_HRSV1B STNKAVVSLSNGVSVLTSKVLDLKNYINNRLLPIVNQQSCRISNIETVTEFQQMNSRLLE ACO83301HRSVA2ref STNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISN1ETVIEFQQKNNRLLE .** **..: :... * : *  ::::*:. : * :.  .*  .:      ::   ..* AAA47881PIV5 IFHNQITNP-ALSPITIQALRILLGSTLPTVVEKSFNTQISAAELLSSGLLTGQIVGLDL FUS_HRSV1B ITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSII ACO83301HRSVA2ref ITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSII * ::  .*. . :*::   *      :* . :  : : :   :. :.     .  :   : AAA47881PIV5 TYMQMVIKIELPTLTVQPATQIIDLATISAFI--NNQEVMAQL--PTRVMVTG--SLTQA FUS_HRSV1B KEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFF ACO83301HRSVA2ref KEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFF .   :.  ::**   *  :.     ::  .    :: . :.           .  *: AAA47881PIV5 YPASQCTITPNTVYCRYNDAQVLSDDTMACLQGN---LTRCTFSPVVGSFLTRFVLFDGI FUS_HRSV1B PQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLG- ACO83301HRSVA2ref PQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLG-   *. *.: .* *:*   :: .*..:.  *  .       *.: .   .. : .:   * AAA47881PIV5 VYANCRS-MLCKCMQPAAVILQPSSSPVTVIDMYKCVSLQLDNLRFTITQLANVTYNSTI FUS_HRSV1B AIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKG ACO83301HRSVA2ref AIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKG . ..* .   *.. :    *::. *.    :.     ::.:.*  : :.:  . .   . AAA47881PIV5 KLESS--QILSIDPLDISQNLAAVNKSLSDALQHLAQSDTYLSAITSATTTS--VLSIIA FUS_HRSV1B EPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTII ACO83301HRSVA2ref EPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTII :   .  : * : . ::. .:: **:.:.::* .: :**  *  :.: .:*:  ::: * AAA47881PIV5 ICLGSLGLILIILLSVVVWKLLTIVVANRNRMENFVYHK--------------------- FUS_HRSV1B IVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK----------------- ACO83301HRSVA2ref IVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN----------------- * :  : * ** :  ::  *  .  *:  :   . : :

Alignment Between F Sequence of HRSV Type A and B with PIV-5 (Top Sequence) that was Used to Construct the Homology Model of Pre-Fusion RSV-F

The other unstable region is the second heptad repeat (HRB) region that forms the trimeric helical stem region in pre-fusion F protein. Deletion of the transmembrane domain (TM) in the soluble F protein further destabilizes this region, which was compensated by the addition of different heterologous trimerization domains. The fully processed mature RSV-F ectodomain was fused C-terminally to different trimerization domains and at different positions (i.e., the F1 domain was truncated at different amino acid residues).

Several constructs were made based on either RSV A2 or B1 strains. Different trimerization domains were linked to the RSV F1 domain, which was truncated at different positions. Trimerization domains that were tested included the Fibritin motif (comprising the amino acid sequence: GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:4), and the “Fibritin long” motif, a longer, N-terminal extended Fibritin domain, which includes its natural helical regions (comprising the amino acid sequence: SSLQGDVQALQEAGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:6), that were added to the RSV F1 domain in frame (in register) with the presumed heptad repeat of the HRB region.

Further constructs that were made comprised heptad ideal helical trimeric coiled coils, or Isoleucine Zipper domains (IZ) (Suzuki et al., Protein Engineering 11:1051-1055 (1998)), comprising the amino acid sequence: IEAIEKK (SEQ ID NO:7). According to the disclosure, different IZ domains were used, referred to as Isoleucine Zipper (L), comprising the amino acid sequence: (I)EKKIEAIEKKIEAIEKKIEAIEAIEKKIEA (SEQ ID NO:8) and Isoleucine Zipper (S), comprising the amino acid sequence EKKIEAIEKKIEAIEKKIEA (SEQ ID NO:3).

These IZ domains are comparable in structure to GCN4, however, the IZ domains are not natural sequences but designed to be optimal trimerization domains and, therefore, more stable.

Further constructs were made with other known trimerization domains:

GCN4II (SEQ ID NO: 9) EDKIEEILSKIYHIENEIARIKKLIGEA Optimized GCN4II (SEQ ID NO: 10) EDKVEELLSKIYHIENRIARIEKLVGEA Matrilin-1 (long version) (SEQ ID NO: 11) EEDPCECKSIVKFQTKVEELINTLQQKLEAVAKRIEALENKII Matrillin-1 short version that only contains zipper domain: (SEQ ID NO: 12) EELINTLQQKLEAVAKRIEALENKII

The following constructs were made:

-   -   Construct F18 comprised the Fibritin trimerization domain (SEQ         ID NO:4) linked to amino acid residue 513 of the F1 domain.     -   Construct F19 comprised the Fibritin trimerization domain (SEQ         ID NO:4) linked to amino acid residue 499 of the F1 domain.     -   Construct F20 comprised the Isoleucine Zipper (L) domain (SEQ ID         NO:8) linked to amino acid residue 516 of the F1 domain and         comprising additional modifications in HRB to optimize the         hydrophobic nature of the heptad positions and facilitate         in-frame fusion with the IZ domain.     -   Construct F21 also comprised Isoleucine Zipper (L) domain (SEQ         ID NO:8), but linked to amino acid residue 501 of the F1 domain         and without additional modifications in the HRB region.     -   Construct F22 comprised the Isoleucine Zipper (L) domain (SEQ ID         NO:8) linked to amino acid residue 495 of the F1 domain and         comprising additional modifications in HRB.     -   Construct F23 comprised the Isoleucine Zipper (S) domain (SEQ ID         NO:3) linked to amino acid residue 495.     -   Construct F46 also comprised the Isoleucine Zipper (S) domain         (SEQ ID NO:3) but linked to a longer RSV-F ectodomain, i.e., the         F1 domain was truncated after amino acid residue 513.

All constructs comprised a HIS-tag.

The constructs were tested for expression levels, storage stability and antibody binding with the antibody CR9501. The amino acid sequences of the heavy and light chain variable regions, and of the heavy and light chain CDRs of this antibody are given below. CR9501 comprises the binding regions of the antibodies referred to as 58C5 in WO2012/006596.

The constructs were synthesized and codon-optimized at Gene Art (Life Technologies, Carlsbad, Calif.). The constructs were cloned into pCDNA2004 or generated by standard methods widely known within the field involving site-directed mutagenesis and PCR and sequenced. The expression platform used was the 293Freestyle cells (Life Technologies). The cells were transiently transfected using 293Fectin (Life Technologies) according to the manufacturer's instructions and cultured for 5 days at 37° C. and 10% CO₂. The culture supernatant was harvested and spun for 5 minutes at 300 g to remove cells and cellular debris. The spun supernatant was subsequently sterile filtered using a 0.22 μm vacuum filter and stored at 4° C. until use.

Supernatants from day 5 were evaluated for F protein expression by Western blot using the monoclonal antibody CR9503, which comprises the heavy and light chain variable regions of the RSV F antibody Motavizumab (referred to as CR9503). The approximate expression levels of the pre-fusion RSV F protein constructs were assessed using CR9503, an anti-human IR-dye conjugated secondary antibody (Li-Cor, Lincoln, Nebr.) or an HRP-conjugated mouse anti-human IgG (Jackson ImmunoResearch, West Grove, Pa.). The protein quantities were then estimated using a dilution series of purified RSV standard protein, either by eye or by using the Odyssey CLx infrared imaging system. Alternatively, Quantitative OCTET® (BioLayer Interferometry) was used for measuring protein concentration in the supernatants. To evaluate construct stability and to identify positive or negative stabilizing effects of introduced trimerization motifs, the constructs capable of binding CR9501 were treated at a range of temperatures from 45-65° C. for 30 minutes to test the stability of the CR9501 epitope. This procedure is described in detail in Example 8. The results are summarized in Table 1.

TABLE 1 Expression and stability of RSV F constructs with different trimerization motifs Description Trimer- Termin- RSV ization ation Expression Protein motif Modifications point (μg/ml) Stability* F18 Fibritin None 513 2 unstable F19 Fibritin None 499 0 ND F20 Isoleucine 502 509 516 516 0 ND zipper (L) Ile F21 Isoleucine None 501 0 ND zipper (L) F22 Isoleucine K483E + 495 0 ND zipper (L) E488K F23 Isoleucine None 495 0.3¹ stable zipper (S) F46 Isoleucine None 513 Did not ND zipper (S) express *Stability is defined as described in Example 7; ND: Not determined. ¹Expression level determined by Western Blot as described in Example 1.

As can be seen in Table 1, the only constructs that were expressed were the Fibritin variant (F18) and F23. Although F18 was trimeric and showed expression, it was unstable upon storage at 4° C. In contrast, F23 was stable at 4° C., binds to the pre-fusion-specific antibodies, but appeared to be monomeric. Therefore, both variants F18 and F23 were used to optimize for both stability and trimerization.

Next, several constructs were made in which the fusion peptide at the N-terminus of F1 was fixed by fusion to the C-terminus of the F2 domain. All constructs comprised a His-tag.

Several constructs were made including constructs in which both furin cleavage sites were mutated resulting in a soluble F protein that still contained the p27 peptide (i.e., F12, F15.1, and F17). In other constructs, the 27-residue region (P27 loop) that is cleaved from the precursor F0 was replaced by an alternative closed loop or linking sequence: either by replacing the region of RSV-F by the “homologous” region of PIV-5 F, the pre-fusion F protein that had been produced and crystallized successfully (F25), or by replacing the region by a minimal (GS)n loop that would bridge the termini of F2 and F1 (F24), or by replacing the region by the central conserved region of RSV-G (F26). Homology modeling of RSV-F based on PIV-5 and in silico measurements resulted in the choice of a minimal loop of five amino acid residues between residues 108 and 136. As a linker, Gly (G) and Ser (S) residues were chosen, which are flexible and polar and have a high chance to be accommodated (F24). Additionally, F137 was mutated to S because the local modifications caused by the loop could displace the hydrophobic F and cause instabilities. This is shown below. Also, the R106 is mutated to Q and 27 residues (109-135) are replaced by GSGSG (SEQ ID NO:5).

(SEQ ID NO: 2) PAANNRARREAPQYMNYTINTTKNLNVSISKKRKRR₁₃₆FLGFLLGVG (SEQ ID NO: 17)

As shown in Table 2, all variants showed no or very low expression except for the variant with the short GSGSG loop (F24), which showed a much higher expression (44 μg/ml) compared to wild-type RSV F construct, i.e., a similar construct, without the linker (F11). F24, which was trimeric, however, was unstable upon storage like all the other variants with a C-terminal Fibritin trimerization motif. All variants contained a HIS-tag.

TABLE 2 Expression and stability of RSV F constructs with different F1-F2 linkers Description RSV Trimerization Termination Expr. Protein Variant motif F1, F2 linker Modifications point (μg/ml) Stability* F11 B1 None None None 513 2.5 stable F18 B1 Fibritin None None 513 2 unstable F12 B1 Fibritin p27 Furin site KO 513 0.1 unstable F15.1 B1 None p27 Furin site KO 525 0.5 ND F17 A2 Fibritin p27 Furin site KO 513 0 ND F24 B1 Fibritin Q_GSGSG_S None 513 44 unstable F25 B1 Fibritin PIV None 513 0 ND F26 B1 Fibritin G CR None 513 0 ND Stability is defined as described in Example 7. Expression level determined as described in Example 1.

Next, the most favorable modifications were combined to find the optimal pre-fusion F polypeptides. Combinations were made of variants with the GSGSG loop, C-terminal truncation of FE and the addition of either fibritin (SEQ ID NO:4) or the Isoleucin Zipper (S) motif (SEQ ID NO:3) (see Table 3).

TABLE 3 Expression and stability of RSV F constructs with combinations of optimizations according to Tables 1 and 2. Stability CR9501 Termi- Description epitope) RSV nation Trimerization F1, F2 Heat Protein Variant point motif linker (μg/ml) (° C.) Storage F11 B1 513 None None  2.5 48 Stable F23 B1 495 Isoleucine None  0.3 ND Stable zipper (S) F24 B1 513 Fibritin Q__GSGSG_S 44   51 Unstable F45 B1 495 Fibritin None  0   ND ND F44 B1 495 Fibritin Q__GSGSG_S  0   ND ND F49 B1 495 None None  2   ND Stable F50 A2 495 None None  2   ND Stable F43 B1 495 Isoleucine Q__GSGSG_S  0.4 53 Stable zipper (S) F47 A2 495 Isoleucine Q__GSGSG_S  5   52 Stable zipper (S) F56 B1 513 Isoleucine Q__GSGSG_S  0.4 ND Stable zipper (S) F46 B1 513 Isoleucine None  0   ND unstable zipper (S) F42 B1 513 None Q__GSGSG S 20   54 Stable F57 A2 513 None Q__GSGSG_S 2-10 54 Stable ND is not determined *Storage stability as determined in Example 7. *Heat stability as determined in Example 8. Expression level as determined by Western blotting (described in Example 1)

Addition of the GSGSG-loop always increased the expression of functional constructs as well as the heat stability of the protein. Combination of the GSGSG-loop with the truncated F and isoleucine zipper (S) motif (F43, F47) showed good expression, heat stability and good stability upon storage at 4° C. However, these variants were still monomeric. The isoleucine zipper (S) trimerization motif showed higher expression with an F variant that was C-terminally truncated F at position 495 (compare F43 with F56 and F23 with F46). In contrast, for variants with the Fibritin trimerization domain, a truncation at position 513 showed high expression compared to truncation at position 495, which showed no expression (compare F24 with F44).

Because the HIS-tag could interfere with the native folding of the trimers, variants were made without the HIS-tag for the Fibritin and the isoleucine zipper (S) variant (Table 4).

TABLE 4 Expression and stability of RSV F constructs with and without HIS-tag Trimer- Termi- Expres- RSV ization F1, F2 nation sion Trimeri- Heat Protein Variant motif linker point μg/ml zation % (° C.) Storage Tags F24 B1 Fibritin Q__GSGSG_S 513 44 Trimeric 51 unstable His-tag (SEC) F24- B1 Fibritin Q__GSGSG_S 513 55 100% ND unstable None (Native) F47 A2 Isoleucine Q__GSGSG_S 495 5   0% 52 stable His-tag zipper (S) (Odyssey) F47- A2 Isoleucine Q__GSGSG_S 495 10 2-5% 53 stable None zipper (S) (Odyssey) A2_F24 A2 Fibritin Q__GSGSG_S 513 5.3 Trimeric 48.75 unstable None (Native) *Storage stability determined as described in Example 7; Heat stability determined as described in Example 8; ND: not determined.

Strikingly, deletion of the HIS-tag increased expression in F47. Moreover, for F47, it increased the trimeric content slightly and for F24, it only increased the expression level moderately.

Next, several alternative trimerization domains and truncations were tested in combination with the GSGSG-loop stabilized F variant (F47) (see Table 5). All variants have a GSGSG-loop and contain a HIS-tag.

TABLE 5 Expression and stability of RSV F variants with alternative trimerization domains Description Trimerization Termination Expression Trimerization Antibody binding RSV Protein Variant motif Modifications point (μg/ml) % CR9501 CR9503 F47 A2 Isoleucine None 495 5 0% + + zipper (S) P1 B1 Isoleucine S502T 502 3.5 0% + + zipper (S) Mat1 A2 Matrillin None 520 12 tri and − + long hexamers Mat2 A2 Matrillin None 516 0 ND − − short Mat3 A2 Matrillin None 495 1.5 ND − − short opt GCN A2 GCN4II None 516 0 ND − − optimized opt A2 GCN4II L512K 516 1 ND + − GCN + L512K optimized Antibody binding is defined as binding on the day of harvest (as described in Example 7); + indicates binding; − indicates no binding. Expression level is determined as described in Example 1. ND: not determined

Only the matrillin 1 domain (Dames-S A et. al., Nat. Struc. Biol., 5(8), 1998) that contains both the N-terminal zipper domain and the C-terminal part with the cysteine residues that can potentially form inter-trimeric disulfide bridges was found to enable higher expression levels than F47 (Table 5, Matrillin long). Moreover, the variant with the Matrillin long trimerization motif shows trimeric F proteins. However, the product did not bind to the pre-fusion-specific Mab CR9501 and also showed hexameric species, which makes the Matrillin 1 trimerization domain not suitable for production of trimeric native F protein. None of the matrillin-based or the GCN4II-based zipper motifs showed increased expression or stability relative to F47 (Table 5, Matrillin short, GCN4II optimized). Again, truncation at 495 results in higher expression levels. Addition of a GCN4 motif, which contained an optimized trigger sequence, showed no expression.

GCN4II is a trimerization domain that is used successfully for stabilizing the pre-fusion trimer of parainfluenza type 5 (Yin et al., Nature 439:38-44, 2006) and has also been tried by others to stabilize RSV pre-fusion F (as disclosed in, e.g., WO2010/149743, WO2010/14975, WO2009/079796, and WO2010/158613). The GCN4II trimerization domain was evaluated and compared with the constructs that contain the Isoleucine Zipper (S) domain (SEQ ID NO:3) or the Fibritin (SEQ ID NO:4) domain (results shown in Table 6). These variants were also compared with other modifications, i.e., a short linker based on a single Lysine and the L512K mutation. All variants contained a HIS-tag.

TABLE 6 Expression and stability of RSV F variants with GCN4II, L512K and p27 replacement (single amino acid linker (K) between F1 and F2) Description Trimer- Termi- Stability RSV ization F1, F2 Modifi- nation Expr. Heat Stor- Protein Variant motif linker cations point (μg/ml) (° C.) age* F18 B1 Fibritin None None 513 2 ND unstable F24 B1 Fibritin Q__GSGSG_S None 513 44 51 unstable F43 B1 Isoleucine Q__GSGSG_S None 495 0.4 53 stable zipper (S) P1 B1 Isoleucine Q__GSGSG_S S502T 502 3.5 54 ND zipper (S) F42 B1 None Q__GSGSG_S None 513 16.1 54 stable P2 B1 None K None 513 14.3 54 stable P3 B1 GCN4II None L512K 516 0 ND ND P4 B1 GCN4II K L512K 516 0 ND ND P5 B1 GCN4II K L512K 516 0 ND ND P6 A2 GCN4II K L512K 516 0 ND ND P7 A2 GCN4II K L512K 516 0 ND ND Storage stability determined as described in Example 7; Expression levels determined as described in Example 1; Heat stability determined as described in Example 8; ND: not determined.

The short linkage between F1 and F2 appears to be comparable to the GSGSG loop. Addition of the GCN4II motif does not result in any F protein expression in any of the constructs (i.e., the RSV A2 F sequence described in WO2010/149743 or WO2010/149745, the RSV A2 F sequence used according to the disclosure, nor the RSV B1 F sequence).

It was shown according to the disclosure that the introduction of these two types of modifications, i.e., introduction of a linking sequence and the heterologous trimerization domain, was not enough to enable the expression of stable trimeric pre-fusion F protein. Apart from the two main regions of instability that were stabilized, i.e., the HRB and the fusion peptide, as described above, other regions in the pre-fusion F protein also contribute and/or accommodate the dramatic refolding to post-fusion F, and more positions in the sequence can be optimized to stop the pre-fusion F protein from refolding. Therefore, different amino acid residues in the HRA and HRB domain and in all domains that contact these regions in pre-fusion F were mutated to increase the pre-fusion structure stability, as described in the following Examples.

Example 2 Preparation of Stable Pre-Fusion RSV F Polypeptides—Stabilizing Mutations

Because the trimeric content (for construct F47) and storage stability (for construct F24) was not optimal, further variants were made that contained point mutations to increase expression levels, stability and native trimeric structure. The results are shown in Tables 7 and 8.

TABLE 7 Expression and stability of F47- variants Expression Trimerization Heat RSV Protein (ug/ml) % (° C.) F47- 10 2-5% 53 F47- + K465E 6 2.4%   ND F47- + D479K 5 29%  50.77 F47- + K176M 13 5% ND F47- + K209Q 9 3% 52.9 F47- + S46G 38 11%  59.38 F47- + S215P 8 1-2% 57.21 F47- + N67I 15 2% 59.84 F47- + K465Q 18 2% 54.3 F47- S46G + N67I 31 6% >60 F47- S46G + S215P 38 6% >60 F47- K465Q + K209Q 12 1% 53.3 F47- K465Q + S46G 28 7% 57.7 F47- K465Q + N67I 17 2% 59 F47- K209Q + N67I 15 4% >60 F47- K209Q + S215P 15 2% 56.7 ND: not determined; Expression level determined as described in Example 1. Heat stability determined as described in Example 8.

Nomenclature of mutations based on wt sequence (SEQ ID NO:1).

All constructs are variants of F47-: type A2, Isoleucine Zipper (S) motif (SEQ ID NO:3), GSGSG linker; termination point 495, no HIS-tag (SEQ ID NO:16). As shown in Table 7, many mutations increased the expression of F47-, but only the variant F47 S46G also showed a higher level of trimers besides the high expression.

Table 8 shows the results of the expression and stability of F24 variants. All variants were of RSV type A2, with fibritin motif, GSGSG linker; termination point 513, no HIS-tag.

TABLE 8 Expression and stability of A2_F24- (SEQ ID NO: 19) variants Expression Storage RSV Protein (μg/ml) Endpoint Association phase A2_F24 5.3 69 ND A2_F24 K508E 5.3 64 ND A2_F24 K498E 1.7 ND ND A2_F24 E487I 25.0 10 ND A2_F24 E487K 7.1 ND ND A2_F24 E487N 42.4 22 ND A2_F24 E487P 12.8 46 ND A2_F24 E487Q* 14.8 50 ND A2_F24 E487R 8.7 59 ND A2_F24 E487S 6.7 46 ND A2_F24 E487Y 10.5 36 ND A2_F24 D486N 31.2 19 ND A2_F24 D479N 5.2 ND ND A2_F24 D479K 1.5 62 ND A2_F24 E472Q 1.9 ND ND A2_F24 E472K 0.9 ND ND A2_F24 K465E 14.8 76 ND A2_F24 K465Q* 13.6 92 Not stable A2_F24 E463K 3.1 ND ND A2_F24 E463Q 6.0 ND ND A2_F24 G430S 4.8 ND ND A2_F24 N428R 5.2 35 ND A2_F24 N426S 18.6 71 ND A2_F24 K421N 9.2 75 ND A2_F24 E328K 9.5 21 ND A2_F24 T311S 3.5 70 ND A2_F24 I309V 11.3 69 ND A2_F24 D269V 0.0 ND ND A2_F24 S215P* 18.7 99 Stable A2_F24 K209Q 31.4 63 ND A2_F24 V207P 3.3 79 ND A2_F24 I206P 5.4 55 ND A2_F24 L204P 5.9 ND ND A2_F24 L203P 0.8 ND ND A2_F24 Q202P 4.4 ND ND A2_F24 K201Q 21.3 62 ND A2_F24 D194P 1.9 ND ND A2_F24 L193P 6.5 42 ND A2_F24 V192P 0.6 32 ND A2_F24 V185N 50.2 38 ND A2_F24 GV184EG 3.5 ND ND A2_F24 G184N 59.8 37 ND A2_F24 V178P 14.8 23 ND A2_F24 A177P 2.0 ND ND A2_F24 K176M 14.7 58 ND A2_F24 K176E 0.7 ND ND A2_F24 N175P 34.3 55 ND A2_F24 S169P 0.5 ND ND A2_F24 K168P 0.1 ND ND A2_F24 K166P 12.3 45 ND A2_F24 V157P 0.2 ND ND A2_F24 E92D 47.4 94 Not stable A2_F24 K85E 1.1 ND ND A2_F24 K80E 51.9 60 ND A2_F24 K77E 22.4 ND ND A2_F24 N67I* 89.8 101  Stable A2_F24 I57V ND ND A2_F24 VI56IV 16.5 54 ND A2_F24 S46G* 40.7 96 Not stable The *marked constructs were tested for trimerization and were all found to be trimeric Expression level determined as described in Example 1. Endpoint stability is shown here as the percentage of pre-fusion antibody binding (CR9501) after 5 days of storage at 4° C. relative to day 1; Association phase stability is determined as described in Example 9.

Many mutations increased the expression of A2_F24-. For most mutations, there was an apparent correlation between improved expression in F47- background (Table 7) and A2_F24- background (Table 8). N67I had more positive impact on F expression in A2_F24− background. The most significant increase in expression was obtained with the single point mutations: S46G, S215P, N67I, K80E, E92D, D486N, G184N, V185N, E487N, N175P, K209Q, E487I, E487Q, K77E, K201Q, N426S and K465Q. In the initial screening using the endpoint stability assay (Example 7), the variants with the highest expression showed the best stability upon storage as well (E92D, K465Q, K465E, N426S, S46G, S215P and N67I). To evaluate if these mutations indeed were stabilizing the pre-fusion conformation, culture supernatants were diluted to 5 and 10 μg/ml based on quantitative Western results and these were stored up to 33 days at 4° C. As single point mutants, only N67I and S215P were completely stable over time (see Example 9).

Subsequently, several mutations that showed high expression and good stability of the pre-fusion conformation were combined to evaluate whether the stabilizations were additive or had a possible synergistic effect (Table 9).

TABLE 9 Expression and stability of variants of A2_F24 with two additional mutations. Expression RSV Protein (μg/ml) stability* A2_F24 K465Q + S46G 21.8 Not stable A2_F24 K465Q + N67I 122.3 Stable A2_F24 K465Q + E92D 10.5 Stable A2_F24 K465Q + S215P 59.8 Stable A2_F24 S46G + N67I 115.5 Stable A2_F24 S46G + E92D 14.3 Not stable A2_F24 N67I + E92D 134.2 Stable A2_F24 N67I + S215P 152.1 Stable A2_F24 E92D + S215P 49.1 Stable A2_F24 K465Q + S215P 53.3 Stable A2_F24 S46G + S215P 43.8 Stable Storage stability refers to the association phase analysis illustrated in Example 9. Expression level was determined as described in Example 1.

All variants are variants of F24-: type A2, fibritin motif, GSGSG linker; termination point 513, binding to all Mabs, no HIS-tag (SEQ ID NO:19).

When the previously identified point mutations were combined, very interesting synergistic effects could be observed, especially in terms of expression levels with the combinations involving N67I as the most potent. All produced double mutants where either N67I or S215P was included and were stable after more than 30 days storage at 4° C. (Example 9). Strikingly, the mutation N67I was found to have the strongest effect on expression levels of pre-fusion F when included in the double mutants. Next, combinations with the S215P mutations resulted in a reasonable expression. Combination of N67I with S215P was selected since it led to a very high expression level, and because both point mutations were stable upon storage. Additionally, it was observed that both N67I and S215P had the ability to stabilize some of the mutants that, as single mutations, were unstable, indicating that the region where these two mutations are found is central for the conformation changes the protein undergoes during the transition to the post-fusion conformation.

According to the disclosure, it thus has been shown that at least some mutations resulted in increased expression levels and increased stabilization of the pre-fusion RSV protein. It is expected that these phenomena are linked. The mutations described in this example all resulted in increased production of pre-fusion F polypeptides. Only a selection of these polypeptides remained stable upon long storage (see Example 9). The stability assay that was used is based on the loss of the pre-fusion-specific CR9501 epitope in the top of the pre-fusion F protein in a binding assay and it may not be sensitive enough to measure all contributions to stability of the whole protein. The mutations for which only increased expression is observed and are, therefore, (very likely stabilizing) potential mutations that can be combined with other stabilizing mutations to obtain a pre-fusion F construct with high stability and high expression levels.

Next, it was verified whether the N67I-S215P double mutation, like the single mutations, was able to stabilize point mutations that as single mutants were deemed unstable based on the criteria used. Extra mutations were selected based on the favorable expression levels and stability according to Table 8. Triple mutant RSV-F variants were constructed and tested for expression levels and stability (Table 10).

TABLE 10 Expression and stability of variants of F24_N67I + S215P with one additional mutation. Expression RSV Protein (μg/ml) stability* A2_F24 N67I + S215P + K507E 344.6 ++ A2_F24 N67I + S215P + E487I 239.4 +++ A2_F24 N67I + S215P + E487N 285.2 +++ A2_F24 N67I + S215P + E487Q 360.7 +++ A2_F24 N67I + S215P + E487R 130.9 +++ A2_F24 N67I + S215P + D486N 292.6 +++ A2_F24 N67I + S215P + D479N 97.1 +++ A2_F24 N67I + S215P + K465Q 283.3 +++ A2_F24 N67I + S215P + N426S 316.3 +++ A2_F24 N67I + S215P + K421N 288.4 +++ A2_F24 N67I + S215P + K209Q 245.0 +++ A2_F24 N67I + S215P + K201Q 231.9 +++ A2_F24 N67I + S215P + V185N 445.1 +++ A2_F24 N67I + S215P + G184N 326.7 +++ A2_F24 N67I + S215P + E92D 308.8 + A2_F24 N67I + S215P + K80E 210.6 + A2_F24 N67I + S215P + S46G 199.4 +++ All variants are variants of A2_F24_N67I + S215P type A2, fibritin motif, GSGSG linker; termination point 513, binding to all Mabs, no HIS-tag (SEQ ID NO: 21). *stability refers to the association phase analysis illustrated in Example 9. + means <10% loss of CR9501 binding after 5 days; ++ means <5% loss of CR9501 binding after 5 days; +++ means 0% loss of CR9501 binding after 5 days.

Again, an additive effect on the expression levels was observed. As expected D479N and E487R triple mutants express at somewhat lower levels because the single mutants were also among the lowest of the selected mutations (Table 8). Because of the stabilizing effect of the N67I+S215P mutation, additional mutations that are unstable as single mutants resulted in stable pre-fusion F variants when they were added to the A2_F24 N67I+S215P background. Some very illustrative examples are the triple mutants with the additional V185N, G184N or E487N, which showed high expression but low stability as single mutants (Table 8) but showed even higher expression and were highly stable when added to the A2_F24 N67I+S215P background.

Stabilizing Mutations Also Stabilize RSV-F Protein from Other Strains and Also in Processed F Variant.

Several mutations that showed high expression and good stability of the pre-fusion conformation were applied to RSV F proteins of other strains (SEQ ID NOS:69 and 70) and were applied to an RSV A2 F variant without furin cleavage site mutations (F18: SEQ ID NO:71) to evaluate whether the modifications are a universal solution to stabilize RSV pre-fusion F (Table 11).

TABLE 11 Expression and stability of variants of A2_F18 with additional mutations and F from strain B1 (SEQ ID NO: 2) and type A CL57-v224 (SEQ ID NO: 69). Relative* Stability** expression after day RSV protein Seq ID (CR9503) 5, % A2_F18 71 0.018 0.0 A2_F18 N67I 0.449 73.2 A2_F18 S215P 0.129 9.1 A2_F18 E487Q 0.006 NA A2_F18 N67I, S215P 72 0.484 103.4 A2_F18 N67I, E487Q 0.340 92.1 A2_F18 N67I, S215P, E487Q 76 0.355 92.7 A2_F18 N67I, S215P, E92D 78 0.318 96.0 A2_F18 N67I, S215P, D486N 79 0.522 101.3 A2_F18 N67I, S215P, K201N 77 0.643 102.7 A2_F18 N67I, S215P, K66E 0.800 103.0 A2_F18 N67I, S215P, S46G, K66E 0.820 103.5 A2_F18 N67I, S215P, E487Q, K66E 0.704 99.5 A2_F18 N67I, S215P, E92D, K66E 0.905 98.8 A2_F18 N67I, S215P, D486N, K66E 0.863 96.6 A2_F18 N67I, S215P, K201N, K66E 1.021 105.5 A2_F18 N67I, S215P, D486N, K66E, 0.594 95.0 I76V B1_N67I, S215P 73 0.434 90.9 B1_N67I, S215P loop 22 0.552 108.2 CL57v224_N67I, S215P 74 0.698 94.9 CL57v224_N67I, S215P loop 75 0.615 98.4 Protein expression (concentration in the supernatant of transiently transfected cells) was measured by Quantitative OCTET ® method. *Relative expression is normalized to expression of A2_F24_N67I, S215P, E487Q (seq ID NO: 33) **Stability - is expressed as % protein concentration measured after storage at 4° C. for 5 days, relative to the day of harvest. The concentrations were measured by Quantitative OCTET ® method using CR9501 antibody. NA—data not available: no CR9501 binding was detected.

When the previously identified point mutations were introduced in A2_F18 (SEQ ID NO:71), the stability and expression levels were very similar compared with the single chain F24 (SEQ ID NO:21) variant that contained a short loop between F1 and F2. Again, synergism was observed showing higher expression and stability when mutations were added to variants that contained the N67I or the double mutation N67I, S215P. The double-point mutation N67I, S215P did not only stabilize the pre-fusion F of the A2 strain but also pre-fusion of the B1 and CL57-v224 strains (Table 11).

Stabilizing Mutations Also Stabilize Full-Length RSV-F Protein.

Several mutations that showed high expression and good stability of the pre-fusion conformation in the soluble version of RSV-F corresponding to the ectodomain were applied to the full-length RSV-F protein. The mutations were introduced in full-length RSV-F with or without furin cleavage site mutations. No trimerization domain was fused to these variants (Table 12)

TABLE 12 Expression and stability of variants of full-length versions of A2_F18 and A2_F24 with additional mutations. Attributes RSV F protein variant* Expression, SEQ F1, F2 fold Heat- Amino acid substitutions ID No linker increase** stability*** None (F A2 wildtype, full length) 1 none 1 - N67I none 1.4 N.D. S215P none 1.4 N.D. E92D none 1.4 N.D. N67I, K465Q none 1.4 N.D. N67I, S46G none 0.2 N.D. N67I, E92D none 1.4 N.D. N67I, K80E none 2.3 N.D. N67I, G184N none 1.5 N.D. N67I, V185N none 1.4 N.D. N67I, E487Q none 2.5 N.D. N67I, S215P, V185N none 2.7 N.D. N67I. S215P, K508E none 3.0 N.D. N67I, S215P, K80E none 3.1 N.D. N67I, S215P, K465Q none 2.9 N.D. N67I, S215P 80 none 2.4 ++ N67I, S215P, G184N none 7.6 ++ N67I, S215P, E92D 82 none 6.8 N.D. N67I, S215P, S46G 88 none 6.8 + N67I, S215P, D486N 86 none 5.9 +++ N67I, S215P, E487Q 84 none 6.2 N.D. N67I, S215P, S46G, K66E none 12.1 +++ N67I, S215P, D486N, K66E none 9.2 +++ N67I, S215P, S46G, E92D, K66E none 11.8 +++ N67I, S215P, S46G, E487Q, K66E none 11.0 +++ N67I, S215P, S46G, D486N, K66E none 10.5 +++ N67I, S215P, D486N, K66E, I76V none 7.2 +++ N67I, S215P, S46G, K66E, I76V none 9.7 +++ N67I, S215P, S46G, K80E none 4.5 N.D. N67I + S215P + G184N + K80E + none 9.1 N.D. E92D + E487Q + S46G None Q__GSGSG_S 3.8 - N67I, S215P 81 Q__GSGSG_S 6.2 N.D. N67I, S215P, G184N Q__GSGSG_S 7.2 ++ N67I, S215P, E92D 83 Q__GSGSG_S 5.9 N.D. N67I, S215P, S46G 89 Q__GSGSG_S 5.3 ++ N67I, S215P, D486N 87 Q__GSGSG_S 5.2 +++ N67I, S215P, E487Q 85 Q__GSGSG_S 4.6 N.D. N67I, S215P, S46G, K66E Q__GSGSG_S 11.7 +++ N67I, S215P, D486N, K66E Q__GSGSG_S 13.8 +++ N67I, S215P, D486N, K66E, I76V Q__GSGSG_S 6.8 +++ N67I + S215P + G184N + K80E + Q__GSGSG_S 3.6 N.D. E92D + E487Q + S46G Expression level determined using FACS. N.D.- not determined. *all variants are based on RSV A2 F protein sequence. **comparing to wild-type protein, fold increase of MFI on 9503. Stability was assessed by heat treatment of the HEK293T cells for 5-10 minutes at 46, 55.3, 60° C. ***legend for the stability readout - decrease in binding to pre fusion-specific Mab CR9501 binding after 46° C. (e.g., wild-type) + slight decrease of CR9501 binding after 46° C., but not to same strong extent as wild-type ++ no change in CR9501 binding up to 60° C.; at 60° C., some decrease in CR9501 binding +++ no change in CR9501 binding at 60° C.

The previously identified stabilizing point mutations were also stabilizing in the full-length F protein. The increase in expression level was less pronounced but showed the same trend. This may be caused by the different background the mutations were introduced in but may also be caused by the different quantification method (FACS versus Western blot) and a biological maximum of expression due to recycling of surface proteins. Introduction of the linking sequence (or short loop) increased expression and stability and the point mutations did so too. The point mutations were not or hardly synergistic with the short loop (similar as to what was found for the soluble protein (Tables 9-11).

Because the point mutation at position 67 had such a positive effect on expression level and stability, all amino acid substitutions were tested for this position to study whether the most optimal were chosen or whether these positions can be improved (Table 13).

TABLE 13 Full substitution analysis of expression and stability for position 67 in the A2_F24 background. Amino acid Relative Stability** after Stability** after day substitution Expression* day 4, % 10, % N67A 1.696 0.0 0.0 N67C 1.759 16.7 0.0 N67D 1.702 0.0 0.0 N67E 1.357 0.0 0.0 N67F 2.565 102.2 108.1 N67G 0.853 NA NA N67H 1.509 0.0 0.0 N67I 3.773 98.2 102.7 N67K 0.487 NA NA N67L 3.609 107.5 96.4 N67M 2.579 87.3 78.7 N67P 2.414 14.3 0.0 N67Q 0.955 NA NA N67R 0.523 NA NA N67S 1.277 0.0 0.0 N67T 1.577 0.0 0.0 N67V 2.457 84.2 77.0 N67W 1.794 99.9 104.3 N67Y 1.830 61.3 45.8 *Relative expression - protein concentration was measured by Quantitative OCTET ® method using CR9503 antibody and expressed relative to concentration of A2_F24 (SEQ ID NO: 19) **Stability - is expressed as % protein concentration measured after storage at 4° C. for 5 and 10 days, relative to the day of harvest. The concentrations were measured by Quantitative OCTET ® method using CR9501 antibody. NA—data not available: no CR9501 binding was detected.

As shown in Table 13, primarily hydrophobic residues and, particularly, Ile, Leu and Met at position 67, were able to increase expression and stability. Ile is the residue that increased expression and stability most. Residues Glu and Gln, the smallest residue Gly, and the positively charged residues Arg and Lys, had the most destabilizing effect at position 67 on the pre-fusion conformation.

According to the disclosure, the amino acid mutations that stabilize the pre-fusion conformation of the RSV F protein can be grouped into different categories that stabilize the conformation in different manners. The strategies for pre-fusion F stabilization are based on the homology model of RSV-F that was based on the PIV-5 crystal structure (Yin et. al., 2006) and the alignment on page 27.

Amino Acid Residues 67 and 215:

The amino acid residues at positions 67 and 215 are very close in the three-dimensional structure of both the pre-fusion model and the post-fusion crystal structure. The residues are close to the conserved disulfide bridge in the top of the DIII region that forms the hinge along which the HRA region refolds into the long-elongated coiled coil-extended helical trimer. Mutations in this region will influence the hinge function and, therefore, the mutations that were introduced stabilize the pre-fusion conformation by obstruction of the hinge function.

Amino Acid Residues 77, 80

The amino acid residues at positions 77 and 80 are located within the long helix (residues 76-98) at the C-terminus of F2 that is in close contact to the ensemble of secondary structures in DIII at the N-terminus of F1 that refold into the long-coiled coil structure of the post-fusion conformation. Since these two regions have to be separated during the refolding from pre- to post-fusion, amino acids in this region that prevent this separation would stabilize the pre-fusion conformation. Because these two regions should part during refolding, some of the residues can be optimized to strengthen the interaction. An example of a repulsion that was observed was between the positively charged Lys80. Mutation of Lys80 to the negatively charged Glu residue increased the expression of pre-fusion F. Due to the sequential transition to the post-fusion conformation, these mutations can be combined with other stabilizing mutations like N67I and S215P to get the full benefit of this stabilization, as shown in Table 10.

Amino Acid Residue 92

The amino acid residue at position 92 is also located within the long helix (residues 76-98) at the C-terminus of F2 that is in close contact to the ensemble of secondary structures in DIII at the N-terminus of F1 that refold into the long-coiled coil structure of the post-fusion conformation. When this helix is separated from the HRA region, it is pulled to the DIII region that contains the Synagis epitope (epitope II) (Arbiza et al., J. Gen. Virol. 73:2225-2234, 1992) and the negatively charged Glu92 moves very close to the positively charged Arg282 in the post-fusion conformation. Mutations that reduce this pull will stabilize the pre-fusion conformation. Mutation of Glu92 to a conserved Asp residue will reduce the pull because it is not able to reach Arg282.

Amino Acid Residues 486, 487

The amino acid residues 486, 487 and 489 at the top of HRB in the pre-fusion conformation form a negatively charged patch mutation of Glu487 to Asn or Ile increased pre-fusion F expression. Mutations of Asp486 into Asn or Gln and/or Glu489 into Asn, Ile or Gln will have the same effect. Due to the sequential transition to post-fusion, these mutations can be combined with other stabilizing mutations like N67I and S215P to get the full benefit of this stabilization, as shown in Table 10 for, e.g., D486N.

Amino Acid Residues 175, 184, 185

In order to refold from the pre-fusion to the post-fusion conformation, the region between residue 175 and 193 has to transform from a loop—beta hairpin to a helix.

This region demonstrates the most dramatic structural transition. Part of this region actually has the highest alpha-helix prediction. The actual helical structures in the pre-fusion model are shown below in grey highlights. This whole region is transformed into one large helix when it refolds to the post-fusion conformation. In the bottom sequence, the residues are highlighted in grey with the highest helix prediction based on Agadir (http://agadir.crg.es/). It is clear from this comparison that the C-terminal part that is maintained in a beta-hairpin in the pre-fusion conformation (residues 187-202) has a high tendency to form an alpha-helix.

The sequence of residues 150-212 of RSV-F (SEQ ID NO:1) is shown above. On the second line, the secondary structures of the top line are indicated by h (for helix) and s (for strands) based on the PIV-5 3D homology model. Helices are highlighted with grey shading. The bottom line is the same sequence in which the helices are shades of grey based on the helix propensity of the sequence.

Therefore, a Proline was introduced at position 175 to stabilize this turn and to prevent refolding into a helix, which, as a single mutation, increased the expression level, indicating that it stabilizes the pre-fusion conformation and enables better processing of the protein. For the turn in the hairpin (residues 184, 185), the Brookhaven database was searched for a structurally homologous hairpin from a stable protein that does not refold. A high structural homology was discovered with a hairpin loop in Protein Kinase A (pdb code 3FHI). According to the alignment shown below, residues 184G1y or 185Val were replaced by Asn in order to stabilize this turn and prevent it from refolding.

HRAb1b2 178-192 (SEQ ID NO: 1) VVSLSNGVSVLTSKV 3fhi:B 179-193 (SEQ ID NO: 97) EMDVYVNNEWATSVG

These mutations can be combined with other stabilizing mutations like N67I and S215P to get the full benefit of this stabilization as shown in Table 10.

Amino Acid Residues 421, 426 and 46

The amino acid residues at positions 421 and 426 are in a loop in the DII region. Residue S46 is on a strand that crosses from DI to DIII. The amino acid residue at position 426 was mutated into serine and the amino acid residue at position 46 was mutated into glycine. These mutations increased stability and pre-fusion expression levels.

Amino Acid Residue 465

The amino acid residue Lys465 is located in another region that goes through a large conformational change. Lys465 is located on a cross-over loop that connects the top of the DII region to HRB. Because the HRB region moves up from the bottom to the top and complexes with HRA to make the six-helix bundle, the cross-over loop also relocates from bottom to top. This loop must, therefore, be metastable in order to allow detachment of DII and reposition in another environment. Lys465 on the cross-over loop is close to Lys445 on the DII region. Mutation of Lys465 to either Gln or Glu neutralizes the repulsion and increased stability and pre-fusion F expression levels.

Example 3 Expression of Pre-Fusion F Protein

Expression plasmids encoding the recombinant pre-fusion RSV F protein were generated by standard methods widely known within the art, involving site-directed mutagenesis and PCR. The expression platform used was the 293Freestyle cells (Life Technologies, Renfreshire, UK). The cells were transiently transfected using 293Fectin (Life Technologies) according to the manufacturer's instructions and cultured in a shaking incubator for 5 days at 37° C. and 10% CO₂. The culture supernatant was harvested and spun for 5 minutes at 300 g to remove cells and cellular debris. The spun supernatant was subsequently sterile filtered using a 0.22 μm vacuum filter and stored at 4° C. until use.

Example 4 Purification of Pre-Fusion RSV F Protein

The recombinant polypeptides were purified by a two-step purification protocol applying a cat-ion exchange column for the initial purification and subsequently a SUPERDEX® 200 column for the polishing step to remove residual contaminants. For the initial ion-exchange step, the culture supernatant was diluted with 2 volumes of 50 mM NaOAc pH 5.0 and passed over a 5 ml HiTrap Capto S column at 5 ml per minute. Subsequently, the column was washed with 10 column volumes (CV) of 20 mM NaOAc, 50 mM NaCl, 0.01% (v/v) TWEEN® 20, pH 5 and eluted 2 CV of 20 mM NaOAc, 1 M NaCl, 0.01% (v/v) TWEEN® 20, pH 5. The eluate was concentrated using a spin concentrator and the protein was further purified using a SUPERDEX® 200 column using 40 mM Tris, 500 mM NaCl, 0.01% (v/v) TWEEN® 20, pH 7.4 as running buffer. In FIG. 1A, the chromatogram from the gel filtration column is shown and the dominant peak contains the pre-fusion RSV F protein. The fractions containing this peak were again pooled and the protein concentration was determined using OD 280 and stored at 4° C. until use. In FIG. 1B, a reduced SDS-PAGE analysis of the final protein preparation is shown and, as can be seen, the purity was >95%. The identity of the band was verified using Western blotting and protein F specific antibodies (not shown).

Example 5 NativePAGE

For initial determination of the multimeric state of the pre-fusion F polypeptides according to the disclosure, culture supernatants from transiently transfected cells were analyzed in a NativePAGE Bis-Tris gel system (Life Technologies). Subsequently, the gels were blotted using the iBlot technology according to the manufacturer's instructions (Life Technologies). An RSV F protein-specific antibody CR9503 (sequences given below in Table 17) was used as primary probe for the detection of pre-fusion RSV F protein and followed by an HRP-conjugated mouse anti-human IgG (Jackson ImmunoResearch, West Grove, Pa.) or an IRDye800CW-conjugated affinity-purified anti-human IgG (rabbit) (Rockland Immunochemicals, Gilbertsville, Pa.). The blots were developed with either standard film (KODAK®) or using the ODYSSEY® CLx infrared imaging system. FIG. 2 shows the NativePAGE analysis of supernatants from monomeric F47- (lane 1), post-fusion and primarily trimeric RSV F protein (lane 2) and purified pre-fusion RSV F protein (lane 3), showing that after purification, only trimeric species are present in the pre-fusion RSV F protein preparation since it migrates similarly to the post-fusion trimer band. This is supported by the elution volume from the gel filtration column as well (FIG. 1A).

Example 6 Quantitative Western Blotting

For quantification of the pre-fusion RSV F protein constructs, quantitative Western blotting was used. Dilutions of culture supernatants were run reduced on 4-12% (w/v) Bis-Tris NuPAGE® gels (Life Technology) and blotted using the IBLOT® technology (Life Technology). The blots were probed with CR9503 (as described above) and developed with either a conjugated mouse anti-human IgG (Jackson ImmunoResearch, West Grove, Pa.) or an IRDye800CW-conjugated affinity-purified anti-human IgG (rabbit) (Rockland Immunochemicals, Gilbertsville, Pa.). The protein quantities were then estimated using a dilution series of purified RSV standard protein and either the ODYSSEY® CLx infrared imaging system or by eye. In FIG. 3, the effects relative to the A2_F24 (SEQ ID NO:19) construct in terms of overall expression levels can be seen. It was shown that single mutations increased the expression level up to five-fold. If double mutants of some of these mutations were generated, synergistic effects could be observed and, in some cases, a further increased expression up to eleven-fold over the A2_F24 was observed.

Example 7 Endpoint Stability Assay

The verification of the pre-fusion conformation of the expressed polypeptides according to the disclosure was done using the BioLayer Interferometry (OCTET®) technology using the pre-fusion-specific antibodies CR9501 or CR9502, or the non-conformation-specific antibody CR9503, which comprises the heavy and light chain variable regions of Motavizumab. The antibodies were biotinylated by standard protocols and immobilized on Streptavidin biosensors (ForteBio, Portsmouth, UK). The procedure was as follows. After equilibration of the sensors in kinetic buffer (FORTEBIO®) for 60 seconds, the Tipss were transferred to PBS with 5 μg/ml of the desired antibody. The loading was carried out for 250 seconds. Subsequently, another equilibration step was included for 200 seconds in kinetic buffer. Lastly, the tipss were transferred to the expression culture supernatant containing the pre-fusion RSV F polypeptides and binding response (nm) after 1200 seconds was recorded. This phase is also referred to as the “association phase.” This was done immediately after harvest (day 1) as well as 5 days later (day 5) and the difference in the CR9501 binding was used as a screening tool to identify mutations capable of stabilizing the pre-fusion conformation. A construct was deemed stable if less than 20% loss of binding was observed at day 5 and if more than 20% loss of binding was observed, it was deemed unstable. Stable constructs could then undergo a more stringent stability test if needed. The data analysis was done using the FORTEBIO® Data Analysis 6.4 software (ForteBio).

Example 8 Heat Stability Assay

The stabilizing potential of introduced features into the RSV F polypeptides was estimated by heat stress. For that purpose, culture supernatant from transiently transfected cells or purified protein was heated using a range of temperatures. The samples were subsequently cooled on ice to prevent further heat-induced conformational changes and probed using the CR9501 antibody on the OCTET® technology platform as described in Example 7. The responses obtained at the end of the association phase at the different temperatures were plotted as a function of the temperature and fitted by non-linear regression using the Prism software. This resulted in an estimation of the temperature where the antibody binding level is 50% of the maximum and this value could be used to compare different constructs in terms of pre-fusion heat stability. In FIGS. 4A and 4B, the unmodified ectodomain (SEQ ID NO:13) and the A2_F24 N67I+S215P construct (SEQ ID NO:21) are compared. It can be observed that temperature-induced stress has less effect on the A2_F24 N67I+S215P construct (SEQ ID NO:21) as compared to the unmodified ectodomain. Therefore, it can be concluded that the stabilizing motifs introduced in the polypeptides according to the disclosure, i.e., the trimerization site, the F1-F2-linker and the two-point mutations, lead to a more stable pre-fusion F protein.

Example 9 Association Phase Stability Assay

To assess the stability of various point mutations, the OCTET® binding assay, a variation of the previously described endpoint stability assay (Example 7), was developed. The association phase analysis was implemented due to the very high expression levels of some point mutants since it is more stringent and completely prevents expression-level bias. The CR9501 antibody was also used, but instead of selecting the binding response at the end of the association phase, the entire association curve was used to reduce potential concentration bias of the endpoint assay. This was done using the data points from the entire association phase of the experiment using the indicated A2_F24 point mutants. The data were compensated for by the amount of bound antibody on the chip. The measurements were done at days 1, 5 and 33, and the shapes of the curves from the three days were compared. If identical curves were obtained, the construct was deemed stable and, if not, unstable. In FIGS. 5A-5E, the analysis of four different variants can be seen. Unstable protein pre-fusion constructs can be identified by a time-dependent loss of CR9501 binding (A2_F24, K465Q, S46G), while stable pre-fusion constructs (N67I) displayed no such decrease. The mutation E92D appeared to fall into a group in between the two, having an intermediate stability since only minor changes in the shape of the curve were observed. In FIGS. 6A-6I, selected point mutations have been combined to make double mutants, and these were analyzed. As can be seen, the different mutations displayed different phenotypes in terms of stability and stability induction. When the polypeptides comprise the mutations K465Q or S46G, alone or in combination, all three, i.e., the two single and the double mutants, are unstable and pre-fusion-specific antibody binding is lost over time. When the mutation S46G is combined with E92D, which previously was shown to have an intermediate stability as a single mutation, no change in stability could be observed, indicating that the E92D mutation cannot correct unstable protein constructs. When the mutation N67I was combined with either the S46G or E92D mutation, the result is a completely stable construct. This could also be observed when the S215P mutation was combined with the E92D mutation showing the unique potential of these two mutations to stabilize unstable pre-fusion constructs.

Example 10 Quantitative OCTET®

To measure concentration of the pre-fusion RSV F protein in cell culture supernatants, the quantitative OCTET®-based method was used. The CR9501 and CR9503 antibodies were biotinylated by standard protocols and immobilized on Streptavidin biosensors (ForteBio, Portsmouth, UK). Afterward, the coated biosensors were blocked in mock cell culture supernatant. Quantitative experiment was performed as follows: temperature 30° C., shaking speed 1000 rpm, time of the assay 300 seconds. Concentration of the protein in the cell culture supernatant was calculated using standard curve. The standard curve was prepared for each coated antibody using the A2_F24_N67I+S215P (SEQ ID NO:21) protein, diluted in mock cell culture supernatant. The measurement was done on the day of supernatant harvest (day 1) and after storage of the supernatant at 4° C. for 5 days or longer. The difference in the concentration determined with the CR9501 was used as a screening tool to identify mutations capable of stabilizing the pre-fusion conformation. A construct was deemed stable if less than 20% decrease of measured concentration was observed at day 5. The data analysis was done using the FORTEBIO® Data Analysis 6.4 software (ForteBio).

Example 11 FACS Analysis and Heat Stability

Expression plasmids encoding the recombinant full-length RSV F protein were generated by standard methods widely known within the art, involving site-directed mutagenesis and PCR. The HEK293-T cells were transiently transfected using 293Fectin (Life Technologies) according to the manufacturer's instructions and cultured for 48 hours at 37° C. and 10% CO₂. The cells were detached from cell culture dishes using FACS® buffer (5 mM EDTA, 1% FBS in PBS), washed and resuspended in the same buffer. Cells were stained for the surface RSV F protein by biotinylated CR9501 or CR9503 antibodies, followed by APC-labeled streptavidin. For discrimination between live and dead cells, propidium iodide was added to the cell suspension at the end of the staining procedure. The cells were analyzed on FACS® Canto (BD Biosciences) according to standard methods well known to any person skilled in the art. The data analysis was done using the FLOWJO® 9.0 software (Tree Star Inc.). Mean fluorescence intensity (MFI) was calculated for the population of live APC-positive cells.

The stabilizing potential of introduced features into the full-length membrane-bound RSV F was estimated by heat stress. Forty-eight hours after transfection, the cells were detached from cell culture dishes as described above and cell suspension was heated for 5-10 minutes using a range of temperatures (37° C., 46° C., 55.3° C., and 60° C.). The cells were subsequently stained and analyzed by FACS® as described above. MFI was calculated for the population of live APC-positive cells. Percent of APC-positive cells was calculated for the live cell population. Staining with the CR9503 resulted in similar MFI and % of APC-positive cells in samples subjected to heat shock with increasing temperatures. Staining with CR9501 was decreasing in cell samples transfected with unstable proteins. Loss of the CR9501 binding indicated loss of the pre-fusion RSV F protein on the cell surface.

Example 12 Preclinical Evaluation of Pre-Fusion F Immunogenicity

To evaluate the immunogenicity of a stabilized pre-fusion RSV F (A2F24, N67I, S215P) (SEQ ID NO:21), mice were immunized according to Table 14 with 0.5 or 5 μg in a prime—boost regimen at week 0 and week 4. As shown in FIG. 7, mice immunized with pre-fusion F showed higher VNA titers than mice immunized with post-fusion RSV F.

TABLE 14 Immunization scheme Group Preparation Dose Adjuvant N 1 Post-fusion F 0.5 μg — 9 2 Post-fusion F   5 μg — 9 3 Pre-fusion F 0.5 μg — 9 4 Pre-fusion F   5 μg — 9 5 Post-fusion F 0.5 μg Poly(I:C) 9 6 Pre-fusion F 0.5 μg Poly(I:C) 9 8 FI-RSV 1/75 — 8 9 PBS — 3

Next, cotton rats were immunized with two different doses of RSV-F in either the post-fusion or the pre-fusion conformation (Table 15). Animals were immunized i.m. at week 0 and week 4. FIG. 8 demonstrates high neutralizing antibody titers at the day of challenge (week 7).

TABLE 15 Groups, immunogen and dose for immunogenicity evaluation and efficacy in cotton rats Group Preparation Dose Adjuvant 1 Post-fusion F 0.5 μg — 2 Post-fusion F   5 μg — 3 Pre-fusion F 0.5 μg — 4 Pre-fusion F   5 μg — 9 Pre-fusion F 0.5 μg Poly IC 10 Pre-fusion F   5 μg Poly IC 11 Pre-fusion F 0.5 μg Adju Phos 12 Pre-fusion F   5 μg Adju Phos 13 Ad26.RSV.F_(A2) 10{circumflex over ( )}8 — 14 PBS — —

Five days after challenge, the lung and nose viral load was measured (see FIG. 9).

As shown, the pre-fusion F polypeptides according to the disclosure are able to induce a strong protective immune response that reduced viral load in the lung and even in the nose.

TABLE 16 Standard amino acids, abbreviations and properties Side chain Side chain charge Amino Acid 3-Letter 1-Letter polarity (pH 7.4) alanine Ala A non-polar Neutral arginine Arg R polar Positive asparagine Asn N polar Neutral aspartic acid Asp D polar Negative cysteine Cys C non-polar Neutral glutamic acid Glu E polar Negative glutamine Gln Q polar Neutral glycine Gly G non-polar Neutral histidine His H polar positive (10%) neutral (90%) isoleucine Ile I non-polar Neutral leucine Leu L non-polar Neutral lysine Lys K polar Positive methionine Met M non-polar Neutral phenylalanine Phe F non-polar Neutral proline Pro P non-polar Neutral serine Ser S polar Neutral threonine Thr T polar Neutral tryptophan Trp W non-polar Neutral tyrosine Tyr Y polar Neutral valine Val V non-polar Neutral

TABLE 17 Antibody sequences Ab VH domain VH CDR1 VH CDR2 VH CDR3 CR9501 Amino acids GASINSDNYYWT HISYTGNTYYTPSLK CGAYVLISNCGWFDS 1-125 of  (SEQ ID NO: 54) S (SEQ ID NO: 56) SEQ ID NO: 53 (SEQ ID NO: 55) CR9502 Amino acids GFTFSGHTIA WVSTNNGNTEYAQKI EWLVMGGFAFDH 1-121 of (SEQ ID NO: 58) QG (SEQ ID NO: 60) SEQ ID NO: 57 (SEQ ID NO: 59) Ab VL domain VL CDR1 VL CDR2 VL CDR3 CR9501 Amino acids QASQDISTYLN GASNLET QQYQYLPYT 1-107 of  (SEQ ID NO: 62) (SEQ ID NO: 63) (SEQ ID NO: 64) SEQ ID NO: 61 CR9502 Amino acids GANNIGSQNVH DDRDRPS QVWDSSRDQAVI 1-110 of (SEQ ID NO: 66) (SEQ ID NO: 67) (SEQ ID NO: 68) SEQ ID NO: 65

The amino acid sequence of several of the pre-fusion RSV F constructs is given below. It is noted that the amino acid numbering in the different constructs described herein is based on the wild-type sequence (SEQ ID NO:1), which means that all amino acids from position 1 to and including position 108 of the pre-fusion constructs correspond to the amino acid positions 1-108 of the wild-type sequence, whereas the numbering of the amino acids from position 138 to the end is shifted 22 amino acids, i.e., L138 in the wild-type sequence (SEQ ID NO:1) corresponds to L116 in all the pre-fusion constructs. This is due to the fact that a deletion has been made in the pre-fusion constructs, i.e., the insertion of the GSGSG linker the actual numbering in F1 is not the same between constructs. Thus, the numbering used with respect to the specific mutations according to the disclosure, e.g., S215P, refers to the position of the amino acid in the wild-type sequence.

Sequences RSV F protein A2 full length sequence (SEQ ID NO: 1) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVT LSKKRKRRFLGELLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVECDTMNSLTLPSEVNLCNV DIENPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTESNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL LHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN RSV F protein B1 full length sequence (SEQ ID NO: 2) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNI KETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVS ISKKRKRRFLGELLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKV LDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSE LLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSP LCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADICKVQSNRVECDTMNSLTLPSEVSLCNT DIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDEL LHNVNTGKSTTNIMITTIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK SEQ ID NO: 3 EKKIEAIEKKIEAIEKKIEA SEQ ID NO: 4 GYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO: 5 GSGSG F8: RSV A2, wt ectodomain (SEQ ID NO: 13) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVT LSKKRKRRELGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNV DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL LHHHHHHHH F11: RSV B1, wt ectodomain (SEQ ID NO: 14) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNI KETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVS ISKKRKRRFLGELLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKV LDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSE LLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSP LCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNT DIENSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDEL LHHHHHHHH F47: RSV A2, linker stabilized, IZ(S) (SEQ ID NO: 15) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI SNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVEKKIEAIEKKIEAIEKKIEAGGIEGRHHHHHHHH F47-: RSV A2, linker stabilized, IZ(S) (SEQ ID NO: 16) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI SNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVEKKIEAIEKKIEAIEKKIEAGG F43: RSV B1, linker stabilized, IZ(S) (SEQ ID NO: 17) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNI KETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNQARGSGSGRSLGFLLGVGSAIAS GIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRI SNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNV QIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWY CDNAGSVSFFPQADICKVQSNRVECDTMNSLTLPSEVSLCNTDIENSKYDCKIMTSKTDISSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVK GEPIINYYDPLVFPSDEFDASISQVEKKIEAIEKKIEAIEKKIEAGGIEGRHHHHHH F24: RSV B1, linker stabilized, fibritin (SEQ ID NO: 18) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNI KETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNQARGSGSGRSLGFLLGVGSAIAS GIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRI SNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNV QIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWY CDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVK GEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGRHHHHHH A2_F24: RSV A2, linker stabilized, fibritin (SEQ ID NO: 19) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI SNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR F24-: RSV B1, linker stabilized, fibritin (SEQ ID NO: 20) NELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNI KETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNQARGSGSGRSLGFLLGVGSAIAS GIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRI SNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNV QIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWY CDNAGSVSFFPQADTCKVQSNRVECDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVK GEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P: A2, linker stabilized, fibritin (SEQ ID NO: 21) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR F24-N67I + S215P: RSV B1, linker stabilized, fibritin (SEQ ID NO: 22) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNI KEIKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNQARGSGSGRSLGELLGVGSAIAS GIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLISKVLDLKNYINNQLLPIVNQQSCRI PNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNV QIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWY CDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVK GEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + E92D: RSV A2, linker stabilized, fibritin (SEQ ID NO: 23) NELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNQARGSGSGRSLGELLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI SNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR F24- N67I + E92D RSV B1, linker stabilized, fibritin (SEQ ID NO: 24) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNI KEIKCNGTDTKVKLIKQELDKYKNAVTDLQLLMQNTPAANNQARGSGSGRSLGFLLGVGSAIAS GIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRI SNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNV QIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWY CDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVK GEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + K465Q RSV A2, linker stabilized, fibritin (SEQ ID NO: 25) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGELLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVITPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDIPCWKLHTSPLCTINTKEGSNICLTRTDRGWY CDNAGSVSEEPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTESNGCDYVSNKGVDTVSVGNTLYYVNKQEGQSLYVK GEPIINFYDPLVEPSDEEDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR F24- N67I + K465Q RSV B1, linker stabilized, fibritin (SEQ ID NO: 26) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNI KEIKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNQARGSGSGRSLGELLGVGSAIAS GIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRI PNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNV QIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWY CDNAGSVSFFPQADTCKVQSNRVECDTMNSLTLPSEVSLCNTDIENSKYDCKIMTSKTDISSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGQNLYVK GEPIINYYDPLVEPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S46G RSV A2, linker stabilized, fibritin (SEQ ID NO: 27) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNQARGSGSGRSLGELLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI SNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVECDTMNSLTLPSEVNLCNVDIENPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTESNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR F24- N67I + S46G RSV B1, linker stabilized, fibritin (SEQ ID NO: 28) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFGALRTGWYTSVITIELSNI KEIKCNGTDTKVKLIKQELDKYKNAVTDLQLLMQNTPAANNQARGSGSGRSLGFLLGVGSAIAS GIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRI SNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNV QIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWY CDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVK GEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 E92D + S215P: A2, linker stabilized, fibritin (SEQ ID NO: 29) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKNKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSINSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR F24-E92D + S215P: RSV B1, linker stabilized, fibritin (SEQ ID NO: 30) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNI KETKCNGTDTKVKLIKQELDKYKNAVT D LQLLMQNTPAANNQARGSGSGRSLGFLLGVGSAIAS GIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRI P NIETVIEFQQKNSRLLEMIREFSVNAGVTTPLSTYMLINSELLSLINDMPIINDQKKLMSSNV QIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWY CDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIENSKYDCKIMTSKTDISSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVK GEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + K508E: A2, linker stabilized, fibritin (SEQ ID NO: 31) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGELLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRE-SDELLSAIGGYIPEAPRDGQAYVRK DGEWVLLSTFLGGIEGR A2_F24 N67I + S215P + E487I: A2, linker stabilized, fibritin (SEQ ID NO: 32) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVEPSDIFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + E487Q: A2, linker stabilized, fibritin (SEQ ID NO: 33) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGELLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDQFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + E487N: A2, linker stabilized, fibritin (SEQ ID NO: 34) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDNFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + D486N: A2, linker stabilized, fibritin (SEQ ID NO: 35) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGELLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTINTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDTFNPKYDCKINTSKTDVSSSV ITSLGAIVSCYGKTKCIASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVEPSNEFDASISQVNEKINQSLAFTRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + K465E: A2, linker stabilized, fibritin (SEQ ID NO: 36) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGELLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGESLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + K465Q: A2, linker stabilized, fibritin (SEQ ID NO: 37) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGQSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + N426S: A2, linker stabilized, fibritin (SEQ ID NO: 38) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGELLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASSKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + K421N: A2, linker stabilized, fibritin (SEQ ID NO: 39) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTNCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + K209Q: A2, linker stabilized, fibritin (SEQ ID NO: 40) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNQQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + K201Q: A2, linker stabilized, fibritin (SEQ ID NO: 41) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDQQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLINSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + V185N: A2, linker stabilized, fibritin (SEQ ID NO: 42) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGELLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGNSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + G184N: A2, linker stabilized, fibritin (SEQ ID NO: 43) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGELLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNNVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVECDTMNSLTLPSEVNLCNVDIENPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTESNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + N175P: A2, linker stabilized, fibritin (SEQ ID NO: 44) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGELLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTPKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLINSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVECDTMNSLTLPSEVNLCNVDIFNPKYDCKINTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + E92D: A2, linker stabilized, fibritin (SEQ ID NO: 45) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNQARGSGSGRSLGELLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + K80E: A2, linker stabilized, fibritin (SEQ ID NO: 46) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIEQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + K77E: A2, linker stabilized, fibritin (SEQ ID NO: 47) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIELIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKINTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24 N67I + S215P + S46G: A2, linker stabilized, fibritin (SEQ ID NO: 48) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24: RSV S46G A2, linker stabilized, fibritin (SEQ ID NO: 49) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNI KKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI SNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24: RSV K465Q A2, linker stabilized, fibritin (SEQ ID NO: 50) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI SNIETVIEFQQKNNRLLEIIREFSVNAGVTTPVSTYMLTNSELLSLIN'MPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVECDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGQSLYVK GEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24: RSV N67I A2, linker stabilized, fibritin (SEQ ID NO: 51) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI SNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR A2_F24: RSV E92D A2, linker stabilized, fibritin (SEQ ID NO: 52) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKNKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI SNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGIEGR RSV F protein CL57-v224 full length sequence (SEQ ID NO: 69) MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANNRARRELPRFMNYTLNNTKNNNVT LSKKRKRRELGELLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNI DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDEL LHNVNVGKSTTNIMITTIIIVIIVILLLLIAVGLFLYCKARSTPVTLSKDQLSGINNIAFSN Ectodomain, RSV CL57-v224 (SEQ ID NO: 70) MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANNRARRELPRFMNYTLNNTKNNNVT LSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNI DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL L PreF, RSV A2, fibritin (SEQ ID NO: 71) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVT LSKKRKRRFLGELLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLINSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTINTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVECDTMNSLTLPSEVNLCNV DIFNPKYDCKINTSKTDVSSSVITSLGAIVSCYGKTKCIASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL LSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL PreF N67I S215P, RSV A2, fibritin (SEQ ID NO: 72) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVT LSKKRKRRELGELLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNV DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDEL LSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL PreF N67I S215P, RSV B1, fibritin (SEQ ID NO: 73) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNI KEIKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVS ISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKV LDLKNYINNQLLPIVNQQSCRIPNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSE LLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSP LCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNT DIENSKYDCKINTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDEL LSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL RSV N67I S215P, RSV CL57-v224, fibritin (SEQ ID NO: 74) MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANNRARRELPRFMNYTLNNTKNNNVT LSKKRKRRELGELLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNI DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDEL LSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL PreFL N67I S215P, RSV B1, fibritin, Loop (SEQ ID NO: 22) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNI KEIKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNQARGSGSGRSLGELLGVGSAIAS GIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRI PNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNV QIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWY CDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVK GEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFL PreFL N67I S215P, RSV CL57-v224, fibritin, Loop (SEQ ID NO: 75) MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANNQARGSGSGRSLGELLGVGSAIAS GIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLINSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFL PreF N67I S215P E487Q, RSV A2, fibritin (SEQ ID NO: 76) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVT LSKKRKRRELGELLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNV DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVEPSDQFDASISQVNEKINQSLAFIRKSDEL LSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL PreF N67I S215P K201N, RSV A2, fibritin (SEQ ID NO: 77) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVT LSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDNQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNV DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL LSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL PreF N67I S215P E92D, RSV A2, fibritin (SEQ ID NO: 78) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVT LSKKRKRRELGELLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNV DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL LSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL PreF N67I S215P D486N, RSV A2, fibritin (SEQ ID NO: 79) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVT LSKKRKRRELGELLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNV DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVEPSNEFDASISQVNEKINQSLAFIRKSDEL LSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL Fwt N67I S215P, membrane-bound RSV F, A2, (SEQ ID NO: 80) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKINVT LSKKRKRRFLGELLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNV DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDEL LHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN Fsl N67I S215P, membrane-bound RSV F, A2, (SEQ ID NO: 81) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVI IVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN Fwt N67I S215P E92D, membrane-bound RSV F, A2, (SEQ ID NO: 82 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVT LSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSINKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNV DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL LHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN Fsl N67I S215P E92D, membrane-bound RSV F, A2, (SEQ ID NO: 83) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVI IVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN Fwt N67I S215P E487Q, membrane-bound RSV F, A2, (SEQ ID NO: 84) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVT LSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVQTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNV DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDQFDASISQVNEKINQSLAFIRKSDEL LHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN Fsl N67I S215P E487Q, membrane-bound RSV F, A2, (SEQ ID NO: 85) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSDQFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVI IVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN Fwt N67I S215P D486N, membrane-bound RSV F, A2, (SEQ ID NO: 86) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVT LSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNV DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVEPSNEFDASISQVNEKINQSLAFIRKSDEL LHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN Fsl N67I S215P D486N, membrane-bound RSV F, A2, (SEQ ID NO: 87) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAvvSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVFPSNEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVI IVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN Fwt N67I S215P S46G, membrane-bound RSV F, A2, (SEQ ID NO: 88) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVT LSKKRKRRFLGELLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKV LDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNV DIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL LHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN Fsl N67I S215P S46G, membrane-bound RSV F, A2, (SEQ ID NO: 89) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNI KKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIAS GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI PNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNV QIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVK GEPIINFYDPLVEPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVI IVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN CR9501 heavy chain (SEQ ID NO: 53): QVQLVQSGPGLVKPSQTLALTCNVSGASINSDNYYWTWIRQRPGGGLEWIGHISYTGNTYYTPS LKSRLSMSLETSQSQFSLRLTSVTAADSAVYFCAACGAYVLISNCGWFDSWGQGTQVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC CR9501 light chain (SEQ ID NO: 61): EIVMTQSPSSLSASIGDRVTITCQASQDISTYLNWYQQKPGQAPRLLIYGASNLETGVPSRFTG SGYGTDFSVTISSLQPEDIATYYCQQYQYLPYTFAPGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC CR9502 heavy chain (SEQ ID NO: 57): EVQLLQSGAELKKPGASVKISCKTSGFTFSGHTIAWVRQAPGQGLEWMGWVSTNNGNTEYAQKI QGRVTMTMDTSTSTVYMELRSLTSDDTAVYFCAREWLVMGGFAFDHWGQGTLLTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC CR9502 light chain (SEQ ID NO: 65): QSVLTQASSVSVAPGQTARITCGANNIGSQNVHWYQQKPGQAPVLVVYDDRDRPSGIPDRFSGS NSGNTATLTISRVEAGDEADYYCQVWDSSRDQAVIFGGGTKLTVLGQPKAAPSVTLFPPSSEEL QANKATLVCLISDPIPGAVTVAWKADSSPVKAGvETTTPSKQSNNKYAASSYLSLTPEQWKSHR SYSCQVTHEGSTVEKTIAPTECS 

1. A recombinant pre-fusion respiratory syncytial virus (RSV) Fusion (F) polypeptide, comprising: at least one epitope that is specific to the pre-fusion conformation F protein, wherein the at least one epitope is recognized by a pre-fusion specific monoclonal antibody comprising: a heavy chain CDR1 region of SEQ ID NO: 54, a heavy chain CDR2 region of SEQ ID NO: 55, a heavy chain CDR3 region of SEQ ID NO: 56, and a light chain CDR1 region of SEQ ID NO: 62, a light chain CDR2 region of SEQ ID NO: 63, and a light chain CDR3 region of SEQ ID NO: 64, and/or a pre-fusion specific monoclonal antibody comprising: a heavy chain CDR1 region of SEQ ID NO: 58, a heavy chain CDR2 region of SEQ ID NO: 59, a heavy chain CDR3 region of SEQ ID NO: 60 and a light chain CDR1 region of SEQ ID NO: 66, a light chain CDR2 region of SEQ ID NO: 67, and a light chain CDR3 region of SEQ ID NO:
 68. 2. Pre-fusion RSV F polypeptide according to claim 1, wherein the polypeptide is trimeric.
 3. The pre-fusion RSV F polypeptide according to claim 1, wherein the polypeptide comprises a mutation of the amino acid residue on position 67 and/or a mutation of the amino acid residue on position
 215. 4. The pre-fusion RSV F polypeptide according to claim 1, wherein the polypeptide comprises a F1 domain and a F2 domain, and a linking sequence comprising from 1 to 10 amino acid residues, linking said F1 domain to said F2 domain.
 5. The pre-fusion RSV F polypeptide of claim 1, comprising a truncated F1 domain and a F2 domain, and a linking sequence comprising 1 to 10 amino acid residues, linking said F1 to said F2 domain, wherein polypeptide comprises at least one stabilizing mutation in the F1 and/or F2 domain as compared to the RSV F1 and/or F2 domain in the wild-type RSV F protein, wherein the at least one stabilizing mutation is selected from the group consisting of: (a) a mutation of amino acid residue N/T on position
 67. (b) a mutation of amino acid residue S on position
 215. 6. The pre-fusion RSV F polypeptide of claim 1, wherein the at least one stabilizing mutation is selected from the group consisting of: (a) a mutation of amino acid residue N/T on position 67 into I; and (b) a mutation of amino acid residue S on position 215 into P.
 7. The pre-fusion RSV F polypeptide according to claim 5, wherein the polypeptide comprises a heterologous trimerization domain linked to said truncated F1 domain.
 8. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide comprises at least one further mutation, wherein said mutation is selected from the group consisting of: (a) a mutation of the amino acid residue on position 46; (b) a mutation of the amino acid residue on position 77; (c) a mutation of the amino acid residue on position 80; (d) a mutation of the amino acid residue on position 92; (e) a mutation of the amino acid residue on position 175; (f) a mutation of the amino acid residue on position 184; (g) a mutation of the amino acid residue on position 185; (h) a mutation of the amino acid residue on position 201; (i) a mutation of the amino acid residue on position 209; (j) a mutation of the amino acid residue on position 421; (k) a mutation of the amino acid residue on position 426; (l) a mutation of the amino acid residue on position 465; (m) a mutation of the amino acid residue on position 486; (n) a mutation of the amino acid residue on position 487; and (o) a mutation of the amino acid residue on position
 508. 9. Pre-fusion RSV F polypeptide according to claim 8, wherein the at least one further mutation is selected from the group consisting of: (a) a mutation of the amino acid residue S on position 46 into G; (b) a mutation of the amino acid residue K on position 77 into E; (c) a mutation of the amino acid residue K on position 80 into E; (d) a mutation of the amino acid residue E on position 92 into D; (e) a mutation of the amino acid residue N on position 175 into P; (f) a mutation of the amino acid residue G on position 184 into N; (g) a mutation of the amino acid residue V on position 185 into N; (h) a mutation of the amino acid residue K on position 201 into Q; (i) a mutation of the amino acid residue K on position 209 into Q; (j) a mutation of the amino acid residue K on position 421 into N; (k) a mutation of the amino acid residue N on position 426 into S; (l) a mutation of the amino acid residue K on position 465 into E or Q; (m) a mutation of the amino acid residue D on position 486 into N; (n) a mutation of the amino acid residue E on position 487 into Q, N or I; and (o) a mutation of the amino acid residue K on position 508 into E.
 10. The pre-fusion RSV F polypeptide according to claim 1, wherein the polypeptide comprises at least two mutations.
 11. Pre-fusion RSV F polypeptide according to claim 10, wherein the at least two mutations comprise a mutation of amino acid residue N/T on position 67 into I; and a mutation of amino acid residue S on position 215 into P.
 12. Pre-fusion RSV F polypeptide according to claim 11, wherein the polypeptide comprises at least one further mutation selected from the group consisting of: (a) a mutation of the amino acid residue S on position 46 into G; (b) a mutation of the amino acid residue K on position 77 into E; (c) a mutation of the amino acid residue K on position 80 into E; (d) a mutation of the amino acid residue E on position 92 into D; (e) a mutation of the amino acid residue N on position 175 into P; (f) a mutation of the amino acid residue G on position 184 into N; (g) a mutation of the amino acid residue V on position 185 into N; (h) a mutation of the amino acid residue K on position 201 into Q; (i) a mutation of the amino acid residue K on position 209 into Q; (j) a mutation of the amino acid residue K on position 421 into N; (k) a mutation of the amino acid residue N on position 426 into S; (l) a mutation of the amino acid residue K on position 465 into E or Q; (m) a mutation of the amino acid residue D on position 486 into N; (n) a mutation of the amino acid residue E on position 487 into Q, N or I; and (o) a mutation of the amino acid residue K on position 508 into E.
 13. The pre-fusion RSV F polypeptide of claim 1, wherein the heterologous trimerization domain comprises SEQ ID NO:3.
 14. Pre-fusion RSV F polypeptide according to claim 13, wherein the trimerization domain is linked to amino acid residue 495 of the RSV F protein.
 15. The pre-fusion RSV F polypeptide of claim 1, wherein the heterologous trimerization domain comprises SEQ ID NO:4.
 16. Pre-fusion RSV F polypeptide according to claim 15, wherein the trimerization domain is linked to amino acid residue 513 of the RSV F protein.
 17. The pre-fusion RSV F polypeptide of claim 1, wherein the linker between the F1 and the F2 domain comprises 5 amino acid residues.
 18. The pre-fusion RSV F polypeptide of claim 17, wherein the linker comprises SEQ ID NO:5.
 19. The pre-fusion RSV F polypeptide of claim 1, wherein the F1 domain and/or the F2 domain are from an RSV A strain.
 20. The pre-fusion RSV F polypeptide of claim 1, wherein the F1 domain and/or the F2 domain are from an RSV B strain.
 21. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide is stable for at least 30 minutes at 55° C.
 22. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide is stable after storage at 4° C. for at least 30 days.
 23. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide comprises a peptide selected from the group consisting of SEQ ID NO: 21-SEQ ID NO: 52 and 71-89.
 24. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide does not comprise a HIS-Tag.
 25. A nucleic acid molecule encoding the pre-fusion RSV F polypeptide of claim
 1. 26. The nucleic acid molecule of claim 25, wherein the nucleic acid molecule has been codon-optimized for expression in mammalian cells.
 27. A vector comprising the nucleic acid molecule of claim
 25. 28. A composition comprising: the pre-fusion RSV F polypeptide of claim 1, and a pharmaceutically acceptable carrier or excipient.
 29. A method of inducing an immune response against RSV F protein in a subject, the method comprising: administering to the subject the pre-fusion RSV F polypeptide of claim 1 in an amount to induce an immune response against RSV F protein in the subject.
 30. A vaccine comprising: the pre-fusion RSV F polypeptide of claim
 1. 31. A method of prophylaxing and/or treating RSV infection in a subject, the method comprising: administering to the subject the pre-fusion RSV F polypeptide of claim 1, to prophylax against and/or treat the subject for RSV infection.
 32. A method for stabilizing the pre-fusion conformation of an RSV F polypeptide, the method comprising: introducing one or more mutations in a RSV F1 and/or F2 domain, as compared to the wild-type RSV F1 and/or F2 domain, wherein the one or more mutations are selected from the group consisting of: (a) a stabilizing mutation in a region that locks the HRA domain from hinging adjacent to the conserved 69-212 disulfide bridge, said region comprising the amino acid residues 66-68 and 214-216, (b) a mutation in the helix comprising the amino acid residues 76-98 at the C-terminus of the F2 domain; (c) a mutation that reduces the negative charge repulsion between the top of the HRB stem region comprising amino acids 486, 487; and (d) a stabilizing mutation in the HRA region.
 33. Method according to claim 32, wherein the mutation in the HRA hinge region is at position
 67. 34. The method according to claim 32, wherein the mutation in the HRA hinge region is at position
 215. 35. The method according to claim 32, wherein the mutation in the HRA hinge region is at position 66 or 68, and/or at position 214 or
 216. 36. Method according to claim 32, wherein the mutation in the helix is at position
 77. 37. Method according to claim 32, wherein the mutation in the helix is at position
 80. 38. The method according to claim 36, wherein the amino acid residue at position 77 and/or 80 is changed into a negatively charged amino acid.
 39. The method according to claim 32, wherein the mutation is at position
 92. 40. Method according to claim 32, wherein the mutation that reduces the negative charge repulsion between the top of the HRB stem region comprising amino acids 486, 487,
 489. 41. Method according to claim 40, wherein the mutation is at position
 489. 42. Method according to claim 40, wherein the mutation is at position
 486. 43. Method according to claim 32, wherein the mutation stabilizes the beta-turns between the amino acid residues 175-193.
 44. Method according to claim 43, wherein the mutation is stabilizing the turn at position
 175. 45. Method according to claim 43, wherein the mutation is stabilizing the turn at position 184-185.
 46. A stabilized pre-fusion RSV F polypeptide, produced by the method according to claim
 32. 47. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide is stable for at least thirty minutes at 58° C.
 48. The pre-fusion RSV F polypeptide of claim 47, wherein the polypeptide is stable for at least thirty minutes at 60° C.
 49. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide is stable after storage at 4° C. for at least sixty days.
 50. The pre-fusion RSV F polypeptide of claim 49, wherein the polypeptide is stable after storage at 4° C. for at least six months.
 51. The pre-fusion RSV F polypeptide of claim 50, wherein the polypeptide is stable after storage at 4° C. for at least one year.
 52. A vector comprising the nucleic acid molecule of claim
 26. 53. The method according to claim 37, wherein the amino acid residue at position 80 is a negatively charged amino acid. 